Pharmaceutical compositions having appetite suppressant activity

ABSTRACT

A pharmaceutical composition contains an extract obtainable from a plant of the genus  Trichocaulon  or  Hoodia  containing an appetite suppressant agent having the formula (1). A process for obtaining the extract and a process for synthesizing compound (1) and its analogues and derivatives is also provided. The invention also extends to the use of such extracts and compound (1) and its analogues for the manufacture of medicaments having appetite suppressant activity. The invention further provides novel intermediates for the synthesis of compound (1)

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 10/073,357,filed Feb. 13, 2002 now abandoned, which is a divisional of applicationSer. No. 09/402,962, filed Oct. 13, 1999, now U.S. Pat. No. 6,376,657,granted Apr. 23, 2002, which is a U.S. National Phase of PCT/GB98/01100,filed Apr. 15, 1998, which claims priority to South African ApplicationNo. 97/3201, filed Apr. 15, 1997, each of which are herein incorporatedby reference in their entireties.

THIS INVENTION relates to steroidal glycosides, to compositionscontaining such steroidal glycosides and to a new use for thesesteroidal glycosides and the compositions containing them. The inventionfurther relates to a method of extracting and isolating these steroidalglycosides from plant material, to a method of synthetically producingthese steroidal glycosides, and to the products of such an extractionand such a synthesis process.

In a particular application, the invention relates to an appetitesuppressant agent, to a process for synthetically producing the appetitesuppressant agent, to a process for extracting the appetite suppressantagent from plant material, to an appetite suppressant compositioncontaining the appetite suppressant agent, and to a method ofsuppressing an appetite.

According to the invention, there is provided a process for preparing anextract of a plant of the genus Trichocaulon or of the genus Hoodia, theextract comprising an appetite suppressant agent, the process includingthe steps of treating collected plant material with a solvent to extracta fraction having appetite suppressant activity, separating theextraction solution from the rest of the plant material, removing thesolvent from the extraction solution and recovering the extract. Theextract so recovered may be further purified, eg by way of suitablesolvent extraction procedures.

The invention also provides a plant extract made of plants of the groupcomprising the genus Trichocaulon and the genus Hoodia and havingappetite suppressant activity.

The extract may be prepared from plant material such as the stems androots of said plants of the genus Trichocaulon or of the genus Hoodia.The genus Trichocaulon and the genus Hoodia include succulent plantsgrowing in arid regions such as are found in Southern Africa. In oneapplication of the invention, the active appetite suppressant extract isobtained from the species Trichocaulon piliferum. The speciesTrichocaulon officinale may also be used to provide an active appetitesuppressant extract. In another application of the invention, the activeappetite suppressant extract may be obtained from the species Hoodiacurrorii, Hoodia gordonii or Hoodia lugardii. Bioassays conducted by theApplicant on rats have indicated that certain of the extracts possessappetite suppressant activity.

The plant material may be homogenised in the presence of a suitablesolvent, for example, a methanol/methylene chloride solvent, by means ofa device such as a Waring blender. The extraction solution may then beseparated from the residual plant material by an appropriate separationprocedure such as, for example, filtration or centrifugation. Thesolvent may be removed by means of the rotary evaporator, preferably ina water bath at a temperature of 60° C. The separated crude extract maythen be further extracted with methylene chloride and water before beingseparated into a methylene chloride extract and a water extract. Themethylene chloride extract may have the solvent removed preferably bymeans of evaporation on a rotary evaporator and the resultant extractmay be further purified by way of a methanol/hexane extraction. Themethanol/hexane extraction product may then be separated to yield amethanol extract and a hexane extract. The methanol extract may beevaporated to remove the solvent in order to yield a partially purifiedactive extract.

The partially purified active extract may be dissolved in methanol, andmay be further fractionated by column chromatography, employing silicagel as an adsorption medium and a chloroform/30% methanol mixture as aneluent. A plurality of different fractions may be obtained, and each maybe evaluated, by suitable bioassaying procedures, to determine theappetite suppressant activity thereof.

A fraction having appetite suppressant activity may preferably befurther fractionated such as by column chromatography using silica gelas an adsorption medium and a 9:1 chloroform:methanol solvent, and theresultant sub-fractions bioassayed for their appetite suppressantactivity. A sub-fraction displaying appetite suppressant activity may,if desired, be further fractionated and purified, conveniently using acolumn chromatographic procedure with silica gel as the adsorptionmedium and a 9:1 ethylacetate:hexane solvent. The resultant purified isfractions may again be evaluated by suitable bioassay procedures fortheir appetite suppressant activity.

The Applicant has found that at least one such purified fraction hasgood appetite suppressant activity, and the active principle in thefraction was identified by conventional chemical techniques includingnuclear magnetic resonance, and was found to be a compound of thestructural formula

In accordance with S.I. nomenclature, the active principle (1) is thecompound3-0-[-β-D-thevetopyranosyl(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl]-12β-0-tigloyloxy-14-hydroxy-14β-pregn-50-en-20-one(C₄₇H₇₄O₁₅M⁺878).

According to another aspect of the invention, there is provided aprocess for preparing an extract of a plant of the genus Trichocaulon orof the genus Hoodia, the extract comprising an appetite suppressantagent, the process including the steps of pressing collected plantmaterial to separate sap from solid plant material and recovering thesap free of the solid plant material to form the extract.

The extract may be dried to remove moisture, e.g. by spray-drying,freeze-drying or vacuum drying, to form a free-flowing powder.

The invention extends to a composition having appetite suppressantactivity comprising an extract as described above.

The composition may be admixed with a pharmaceutical excipient, diluentor carrier and optionally it is prepared in unit dosage form.

The invention also extends to the use of an extract as described abovein the manufacture of a medicament having appetite suppressant activity,to an extract as described above for use as a medicament having appetitesuppressant activity, and to a method of suppressing an appetite byadministering to a human or animal an effective dosage of a compositionas described above.

Compound (1) is a novel compound and the invention extends to compound(1) and certain analogues or derivatives of this steroidal trisaccharidehaving appetite suppressant properties. The molecules chosen as theanalogues or derivatives are intended to affect the properties of thesteroidal trisaccharide with the aim of increasing the activity of theactive ingredient. The following effects were taken into considerationwhen the analogues were chosen:

-   (i) Hydrophobic interactions and lipophilicity    -   Functional group modifications of the active molecule is        intended to change the hydrophobicity and lipophilicity of the        molecule. Increased lipophilicity has been shown to correlate        with increased biological activity, poorer aqueous solubility,        increased detergency/cell lysis, increased storage in tissues,        more rapid metabolism and elimination, increased plasma protein        binding and faster rate of onset of action.-   (ii) Electronic properties and ionization constants    -   Functional group modification of the molecule is also intended        to change the acidity and basicity which would have a major role        in controlling the transport of the compound to its site of        action and the binding at this target site.-   (iii) Hydrogen bonding    -   Functional group modifications of carboxyl and carbonyl groups        in the active molecule are intended to change the interactions        between the proteins in biological systems and the chemically        modified functional groups.-   (iv) Steric parameters    -   The purpose of changing the steric features of the molecule is        to increase binding to its receptor and thus increase its        biological activity.        The following chemical modifications to the molecule are        intended to affect the hydrophobicity and lipophilicity        electronic properties, hydrogen bonding and steric parameters on        the molecule:-   a) Chemical modification of the C-12 group and ester functionality;-   b) Chemical modification of the 5,6-double bond, e.g. hydrogenation    and migration;-   c) Chemical modification of the C-20 carbonyl and C-17 acetyl group;-   d) Chemical modification of the “D” ring of the steroid or aglycone    ring;-   e) Modification of the carbohydrates of the trisaccharide moiety.

Accordingly, the invention provides a compound having the generalstructural formula

in which R=alkyl;

-   -   R₁=H, alkyl, tigloyl, benzoyl, or any other organic ester group;    -   R₂=H, or one or more 6-deoxy carbohydrates, or one or more        2,6-dideoxy carbohydrates, or glucose molecules, or combinations        thereof;    -   and in which the broken lines indicate the optional presence of        a further bond between C4–C5 or C5–C6.

The invention also provides a compound as described above wherein thereis a further bond between C5-C6, R=methyl, R₁=tigloyl,R₂=3-0-([-β-D-thevetopyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl]and having the structural formula.

Further active analogues or derivatives of the appetite suppressantcompound (1) in accordance with the invention are compounds having thefollowing structural formulae:

in which R=alkyl; and

-   -   R₁=H, or benzoyl, or tigloyl, or any other organic ester group

in which R=alkyl; and

-   -   R₁=H, or tigloyl, or benzoyl, or any other organic ester group

in which R=alkyl; and

-   -   R₁=H, or tigloyl, or benzoyl, or any other organic ester group

in which R=alkyl; and

-   -   R₁=H, or tigloyl, or benzoyl, or any other organic ester group

in which R=alkyl;

-   -   R₁=H, or tigloyl, or benzoyl, or any other organic ester group.

in which R=alkyl; and

-   -   R₁=H, alkyl, tigloyl, benzoyl, or any other organic ester group;    -   R₂=H, or one or more 6-deoxy carbohydrates, or one or more        2,6-dideoxy carbohydrates, or glucose molecules, or combinations        thereof;    -   and in which the broken lines indicate the optional presence of        a further bond between C4–C5 or C5–C6.

in which R=alkyl; and

-   -   R₁=H, alkyl, tigloyl, benzoyl, or any other organic ester group;    -   R₂=H, or one or more 6-deoxy carbohydrates, or one or more        2,6-dideoxy carbohydrates, or glucose molecules, or combinations        thereof;    -   and in which the broken lines indicate the presence of a further        bond between C4–C5 or C5–C6.

in which R=alkyl; and

-   -   R₁=H, alkyl, tigloyl, benzoyl, or any other organic ester group;    -   R₂=H, or one or more 6-deoxy carbohydrates, or one or more        2,6-dideoxy carbohydrates, or glucose molecules, or combinations        thereof;    -   and in which the broken lines indicate the optional presence of        a further bond between C4–C5 or C5–C6.

in which R=alkyl; and

-   -   R₁=H, alkyl, tigloyl, benzoyl, or any other organic ester group;    -   R₂=H, or one or more 6-deoxy carbohydrates, or one or more        2,6-dideoxy carbohydrates, or glucose molecules, or combinations        thereof;    -   and in which the broken lines indicate the optional presence of        a further bond between C4–C5, C5–C6 or C14–C15.

in which R=alkyl; and

-   -   R₁=H, alkyl, tigloyl, benzoyl, any other organic ester group;    -   R₂=H, or one or more 6-deoxy carbohydrates, or one or more        2,6-dideoxy carbohydrates, or glucose molecules, or combinations        thereof;    -   and in which the broken lines indicate the optional presence of        a further bond between C4–C5, C5–C6 or C14–C15.

in which R=alkyl; and

-   -   R₁=H, alkyl, tigloyl, benzoyl, any other organic ester group;    -   R₂=H, or one or more 6-deoxy carbohydrates, or one or more        2,6-dideoxy carbohydrates, or glucose molecules, or combinations        thereof;    -   and in which the broken lines indicate the optional presence of        a further bond between C4–C5, C5–C6 or C14–C15; and    -   R₃=H, alkyl, aryl, acyl, or glucoxy.

in which R=H, alkyl, aryl or any steroid possessing a C14 beta hydroxygroup, or a C12 beta hydroxy functionality, or a C17 acyl group, or aC5–C6 olefin, or combinations thereof.

The invention still further extends to a process for syntheticallyproducing a compound having appetite suppressant activity.

The process uses a steroid as a starting material (or intermediate orprecursor) the steroid having the chemical formula

The steroid (15) can be prepared from a compound having the formula (22)by a process which includes the steps of

-   (i) treating progesterone having the formula

with the micro-organism Calonectria decora to produce a compound 12β,15α-dihydroxy progesterone of the formula

-   (ii) treating compound 17) with tosyl chloride and pyridine to    produce a compound 12β-hydroxy-15α-(p-toluene sulfonyl)-progesterone    of the formula

-   (iii) treating the compound (18) with collidine at 150° C. to    produce a compound 12β-hydroxy-Δ¹⁴-progesterone of the formula

-   (iv) treating the compound (19) with acetyl chloride and acetic    anhydride at 120° C., to produce a compound    3,12β-diacetoxypregna-3,5,14-trien-20-one of the formula

-   (v) treating the compound (20) with ethylene glycol and a catalytic    amount of p-toluene sulphonic acid, to produce a compound    3,12β-diacetoxy-20,20-ethylenedioxypregna-3,5,14-triene of the    formula

-   (vi) treating the compound (21) with NaBH₄ to produce a compound    3β,12β-dihydroxy-20,20-ethylenedioxypregna-5,14-diene-12-acetate of    the formula

In a first alternative procedure, a process for the preparation ofsteroid (15) according to the invention includes the steps of

-   (a) treating compound (22) with a reducing agent, e.g. LiAlH₄, to    produce a compound 3β, 12β-dihydroxy-20,20-ethylenedioxypregna-5,    14-diene of the formula

-   (b) treating compound (23) with N-bromoacetamide (NBA) and a base,    e.g. pyridine, to produce a compound 3β, 12β-dihydroxy-14,    15-epoxy-20,20-ethylenedioxypregn-5-ene of the formula

-   (c) treating compound (24) with a reducing agent, e.g. LiAlH₄, e.g.    with refluxing, to produce a compound 3β, 12β,    14β-trihydroxy-20,20-ethylenedioxypregn-5-ene of the formula

-   and (d) treating compound (25) with an acid, e.g. acetic acid, and    water to produce the steroid intermediate compound 3β, 12β,    14β-trihydroxy-pregn-5-ene (15).

Reaction Scheme A depicts the procedure for the preparation of steroidintermediate (15) from compound (22) according to “the first alternativeprocedure” of the invention (and includes the preparation of compound(22) from compound (16) for illustrative purposes).

In a second alternative procedure, a process for the preparation ofsteroid (15) according to the invention includes the steps of

-   (a) treating compound (22) (3β,    12β-dihydroxy-20,20-ethylenedioxypregna-5,14-diene-12-acetate) with    p-toluenesulfonyl chloride and a base, e.g. pyridine, to produce a    compound    3β,12β-dihydroxy-20,20-ethylenedioxypregna-5,14-diene-3-tosyl-12-acetate    of the formula

-   (b) treating compound (26) with potassium acetate in a solvent, e.g.    acetone, to produce a compound 6β,    12β-dihydroxy-20,20-ethylenedioxy-3,5α-cyclopregnan-14-ene-12-acetate    of the formula

-   (c) treating the compound (27) with a reducing agent, e.g. LiAlH₄,    and e.g. tetrahydrofuran, to produce a compound 6β,    12β-dihydroxy-20,20-ethylenedioxy-3,5α-cyclopregnan-14-ene of the    formula

-   (d) treating the compound (28) with N-bromoacetamide, optionally    acetic acid, and a base, e.g. pyridine, to produce a compound 6β,    12β-dihydroxy-20,20-ethylenedioxy-14,15-epoxy-3,5α-cyclopregnane of    the formula

-   (e) treating the compound (29) with a reducing agent, e.g. LiAlH₄,    and e.g. tetrahydrofuran, to produce a compound 6β, 12β,    14β-trihydroxy-20,20-ethylenedioxy-3,5α-cyclopregnane of the formula

-   and (f) treating compound (30) with an acid, e.g. hydrochloric acid,    and a solvent e.g. acetone, to produce compound (15).

Reaction Scheme B shows the procedure for the preparation of steroidintermediate (15) from compound (22) according to “the secondalternative procedure” of the invention.

Compound (1) may be synthesized from a first carbohydrate intermediatein the form of an activated monosaccharide cymarose moiety, which can beprepared from a compound having the formula (36). Compound (36) can beprepared by a process which includes the steps

-   (i) treating methyl-α-D-glucose having the formula

-    with benzaldehyde and zinc chloride to produce a compound    methyl-4,6-0-benzylidene-α-D-glucopyranoside of the formula

-   (ii) treating the compound (32) with tosyl chloride and pyridine at    0° C., to produce a compound    methyl-4,6-0-benzylidene-2-0-tosyl-α-D-glucopyranoside of the    formula

-   (iii) treating the compound (33) with NaOMe at 100° C. to produce a    compound methyl 4,6-0-benzylidene-3-0-methyl-α-D-altropyranoside of    the formula

-   (iv) treating the compound (34) with N-bromosuccinamide (NBS) to    produce a compound methyl    6-bromo-4-0-benzoyl-3-0-methyl-6-deoxy-α-D-altropyranoside of the    formula

-   and (v) treating the compound (35) with NaBH₄ and NiCl₂, to produce    a compound methyl 4-0-benzoyl-3-0-methyl-6-deoxy-α-D-altropyranoside    of the formula

The invention extends to a process for the preparation of a carbohydrateintermediate in the form of an activated monosaccharide cymarose moietywhich includes the steps of

-   (i) treating the compound (36) with PhSSiMe₃, ZnI₂ and Bu4⁺I⁻ to    produce a compound    4-0-benzoyl-3-0-methyl-6-deoxy-αβ-D-phenylthioaltroside of the    formula

-   (ii) optionally treating the compound (37) with diethylaminosulphur    trifluoride (DAST), e.g. at 0° C., to produce a compound    4-0-benzoyl-3-0-methyl-2-phenylthio-2,6-dideoxy-αβ-D-fluorocymaropyranoside    having the formula

-   or (iii) optionally, treating the compound (37) with    t-butyldimethylsilylchloride and imidazole in a solvent, e.g.    pyridine, to produce    4-0-benzoyl-3-0-methyl-2-0-t-butyldimethylsilyl-αβ-D-phenylthioaltroside    having the formula

-    in which Z=TBDMS=t-butyldimethylsilyl-   and (iv) treating the compound (39) with a base, e.g. sodium    methoxide, to produce    3-0-methyl-2-0-t-butyldimethylsilyl-αβ-D-phenylthioaltroside having    the formula

-    in which Z=TBDMS=t-butyldimethylsilyl.

Reaction Scheme C shows the procedure for the synthesis of the activatedmonosaccharide cymarose moiety (40) from compound (36) according to theinvention (and includes the preparation of compound (36) from compound(31) for illustrative purposes).

The synthesis of compound (1) may also involve a second carbohydrateintermediate in the form of an activated monosaccharide thevetosemoiety, which can be prepared from a compound having the formula (47).Compound (47) can be prepared by a process which includes the steps of

-   (i) treating α-D-glucose having the formula

-    with acetone and sulphuric acid to produce a compound 1,2:    5,6-di-0-isopropylidene-α-D-glucofuranose of the formula

-   (ii) treating the compound (42) with NaH and MeI to produce a    compound 1,2:5,6-Di-0-isopropylidene-3-0-methyl-α-D-glucofuranose of    the formula

-   (iii) treating the compound (43) with acetic acid to produce a    compound 3-0-methyl-αβ-D-glucopyranose of the formula

-   (iv) treating the compound (44) with methanol and hydrochloric acid    to produce a compound methyl 3-0-methyl-αβ-D-glucopyranoside having    the formula

-   (v) treating the compound (45) with benzaldehyde and zinc chloride    to produce a compound methyl    4,6-0-benzylidene-3-0-methyl-αβ-glucopyranoside having the formula

-   (vi) treating the compound (46) with N-bromosuccinamide, nickel    chloride and sodium borohydride to produce a compound methyl    4-0-benzoyl-3-0-methyl-6-deoxy-αβ-glucopyranoside having the formula

The invention extends to a process for the preparation of an activatedmonosaccharide thevetose moiety which includes the steps of

-   (i) treating the compound (47) with phenylthiotrimethysilane and    trimethylsilyltrifluoromethanesulphonate to produce a compound    4-0-benzoyl-3-0-methyl-1-phenylthio-6-deoxy-αβ-glucopyranoside    having the formula

-   (ii) treating the compound (48) with pivaloyl chloride and a    solvent, e.g. pyridine, to produce a compound    4-0-benzoyl-3-0-methyl-2-0-pivaloyl-1-phenylthio-6-deoxy-αβ-glucopyranoside    having the formula

-   and (iii) treating the compound (49) with a brominating agent, e.g.    N-bromosuccinimide, and diethylaminosulphur trifluoride to produce a    compound    4-0-benzoyl-3-0-methyl-2-0-pivaloyl-1-fluoro-6-deoxy-β-glucopyranoside    occurring as stereo-isomers having the formula

Reaction Scheme D shows the procedure for the synthesis of the activatedmonosaccharide thevetose moiety (50(A) and 50(B)) from compound (48)according to the invention (and includes the preparation of compound(47) from compound (41) for illustrative purposes).

According to a still further aspect of the invention there is provided aprocess of synthetically producing a compound of the formula (1) andanalogues and derivatives thereof which includes the steps ofsynthesising a suitable steroid intermediate or precursor and couplingthe required number of suitable monosaccharides with the steroidintermediate.

The invention also provides a process of coupling a monosaccharidecymarose with the steroid intermediate, which includes the steps of

-   (i) reacting a cymarose moiety (38) with a steroid intermediate    (15), e.g. at −15° C., and in the presence of tin chloride, in a    solvent, e.g. ether, to produce a compound    3-0-[4-0-benzoyl-2-phenylthio-β-D-cymaropyranosyl]-12,14-β-dihydroxy-pregn-5-ene-20-one    of the formula

-   and (ii) treating the compound (51) with tiglic acid chloride in    pyridine and thereafter with a base, e.g. NaOMe, to produce a    compound 3-0-[-2-phenylthio-β-D-cymaropyranosyl]-12β-tigloyloxy-    14-hydroxy-14β-pregn-5-ene-20-one of the formula

The invention extends to a process which includes coupling amonosaccharide cymarose moiety to a monosaccharide thevetose moiety andcoupling the resultant disaccharide with the combined steroid product(52) to form compound (1).

The process of coupling the monosaccharide cymarose moiety to themonosaccharide thevetose moiety and coupling the resultant disaccharideto the combined steroid product (52) may include the steps of

-   (i) coupling a selectively protected cymarose moiety (40) and a    selectively protected thevetose moiety (50 A) using tin chloride    (SnCl₂) and silver trifluoromethanesulphonate, e.g. at −15° C., to    produce a compound of the formula

-    in which Z=TBDMS=t-butyldimethylsilyl-   (ii) treating compound (53) with tetrabutylammoniumfluoride to    produce a compound of the formula

-   (iii) treating compound (54) with diethylaminosulphur trifluoride,    e.g. at 0° C., to produce a compound of the formula

-   (iv) reacting compound (55) with compound (52) to produce a compound    of the formula

-   and (v) treating compound (56) in a Raney-Nickel reaction and    thereafter with a base, e.g. NaOMe, to produce compound (1) as    described above.    Reaction Scheme E shows the procedure for the synthesis of    intermediates (52) and (55) and coupling them to form compound (56).

According to the invention, an alternative process is provided whichincludes coupling cymarose and thevetose moieties to form atrisaccharide and coupling the trisaccharide onto a steroid derivativeto form a compound of the formula (1).

The process of forming the trisaccharide and coupling the resultanttrisaccharide to a steroid derivative may include the steps of

-   (i) coupling a selectively protected cymarose moiety (40) and    compound (45) using tin (II) chloride, AgOTf, Cp₂ZrCl₂ to produce a    compound of the formula

in which Z=TBDMS=t-butyldimethylsilyl

-   (ii) treating compound (57) with tetrabutylammoniumfluoride and    diethylaminosulphur trifluoride to produce a trisaccharide compound    having the formula

-   and (iii) coupling the trisaccharide (58) with a steroid    intermediate of the formula

-   -   using tin (II) chloride, AgOTf, Cp₂ZrCl₂ to produce compound        (1).

The steroid intermediate (59) may be produced by treating steroid (15)with tiglic acid chloride.

Reaction Scheme F shows the procedure for the synthesis of thetrisaccharide (58) and the synthesis of compound (1) by coupling thetrisaccharide (58) with the steroid intermediate (59).

The intermediates (23), (24), (25), (27), (28), (29), (30), (37), (38),(39), (40), (48), (49), (50), (51), (53), (54), (55), (56), (57) and(58) described above are novel compounds and the invention extends tothese compounds as such.

Compound (1),3-0-[-β-D-thevetopyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl]-12β-0-tigloyloxy-14-hydroxy-14β-pregn-5-en-20-one,and various analogues and derivatives thereof have been found to haveappetite suppressing activity.

The invention extends also to a composition or formulation havingappetite suppressant activity, in which the active ingredient is anextract obtained from a plant of the genus Trichocaulon or the genusHoodia.

The active ingredient may be a compound of the formula (1), extractedfrom a plant of the genus Trichocaulon or Hoodia or a derivativethereof. The plant may be of the species Trichocaulon officinale orTrichocaulon piliferum, or the species Hoodia currorii, Hoodia gordoniior Hoodia lugardii.

The invention extends also to a composition or formulation havingappetite suppressant activity, in which the active ingredient is asynthetically produced compound of the formula (1) or a derivative oranalogue thereof, as hereinbefore set out with reference to compounds(2) to (14).

According to another aspect of the invention there is provided a methodof suppressing an appetite by administering to a human or animal asuitable dosage of an appetite suppressant agent comprising an extractof a plant of the genus Trichocaulon or Hoodia. The extract may beincorporated in a composition or formulation including alsopharmaceutically acceptable other ingredients.

The appetite suppressant agent may be an isolated natural chemical or asynthetic chemical compound of the formula:

or derivatives or analogues thereof, as set out before.

The appetite suppressant composition or formulation may consist of theappetite suppressant agent admixed with a pharmaceutical excipient,diluent or carrier. Other suitable additives, including a stabilizer andsuch other ingredients as may be desired may be added.

The invention extends to the use of compound (1) or its derivatives oranalogues in the manufacture of a medicament having appetite suppressantactivity.

The invention further extends to compound (1), or its derivatives oranalogues as set out before, for use as a medicament having appetitesuppressant activity.

A method of suppressing an appetite by administering to a human oranimal an effective dosage of a composition as described above is alsoprovided.

A method has been described herein for extracting a steroidal glycosidehaving appetite suppressant activity from plant material obtained from aplant of the Trichocaulon or Hoodia genus. The invention thus extends toan extract obtained from plant material of the Trichocaulon or Hoodiagenus and containing a substantially pure steroidal glycoside of formula(1).

The invention extends also to a foodstuff or a beverage containing aneffective quantity of the steroidal glycoside of the formula (1), or itsderivatives or analogues as set out before, to have an appetitesuppressant effect when ingested.

Molecular genetic studies have led to a considerable increase in theunderstanding of the regulation of appetite, satiety and bodyweight.These studies have revealed numerous central regulatory pathways,mediated by a number of neuropeptides. The maintenance of a normal bodyweight is achieved by an intricate balance between energy intake, foodconsumption, and energy expenditure. Energy homeostasis is subject to awide range of influences, ultimately controlled by the brain. Thedifferent signals include such things as sense of smell and taste andgastrointestinal signals such as distension of the gastrointestinaltract, chemical signals to the gastric mucosa and blood-bornemetabolites such as fatty acids and glucose.

Centrally, neuropeptide “Y” (NPY) which is negatively regulated byleptin, has been established as one of the positive regulators offeeding behaviour. Expression of the endogenous antagonist formelanocortin receptors has also been shown to be the basis for obesityin a particular model (the ob/ob mouse). Indeed deficiency at the MC4melanocortin receptor completely replicates the obesity syndrome. Othermediators which have been shown to have roles in the energy balanceinclude bombesin, galonin and glucagon-like peptide-1.

Without being bound by theory, the Applicant believes that compound (1)and its analogues as described above act as an agonist of themelanocortin 4 receptor. The effect of this is to regulate NPY but alsoto increase cholecystokinin. The effect of cholecystokinin amongst otherthings is to inhibit gastric emptying.

Accordingly, the invention extends to a composition having appetitesuppressant activity comprising a melanocortin 4 receptor agonist.

The agonist may be an extract or compound as previously described, inparticular the compound of formula (1). The composition may be admixedwith a pharmaceutical excipient, diluent or carrier and is optionallyprepared in unit dosage form.

The invention still further extends to the use of a melanocortin 4receptor agonist in the manufacture of a medicament having appetitesuppressant activity, to a melanocortin 4 receptor agonist for use as amedicament having appetite suppressant activity, to a method ofsuppressing an appetite by administering to a human or animal aneffective dosage of a composition comprising a melanocortin 4 agonist asdescribed above, and to the use of a melanocortin 4 receptor agonist tosuppress the appetite of and/or to combat obesity in a human or animal.

The invention and its efficacy will now be further described, withoutlimitation of the scope of the invention, with reference to thefollowing examples and drawings.

In the drawings,

FIG. 1 shows a flow diagram of the general method of extracting a firstcrude appetite suppressant extract and a purified appetite suppressantextract from plant material of the genus Trichocaulon or Hoodia;

FIG. 2 shows a graphical representation of a bioassay carried out onrats using a partially purified methanol extract of Trichocaulonpiliferum;

FIGS. 3 and 4 together show a schematic representation of a preferredembodiment of the process of the invention for producing an extract ofplant material of the genus Trichocaulon or Hoodia; and

FIGS. 5 and 6 show a graphical representation of the percentage changeof body mass of rats for different groups for days −7 to 7 and days 0 to7 respectively in a repeat dose study using a sap extract and aspray-dried sap extract of plant material of the species Hoodiagordonii.

EXAMPLE 1

The general method of extracting a first crude appetite suppressantextract and a purified appetite suppressant extract from plant materialof the genus Trichocaulon or of the genus Hoodia is illustrated by wayof the flow diagram of FIG. 1.

EXAMPLE 2

Bioassays carried out on rats using a partially purified methanolextract obtained in the manner illustrated in Example 1, indicated thatthe extract does in fact exhibit appetite suppressant activity. Theappetite suppressant activity of the active extract can be illustratedby way of a typical example of the effect of the methanol extract ofTrichocaulon piliferum on rats, by way of the graphic representation inFIG. 2.

It will be evident from FIG. 2 that the test group of rats dosed withthe extract on day 5 displayed a substantially diminished food intakeover the next two days, while a control group did not disclose acomparable reduced food intake. The food intake of the test groupreturned to normal, and in fact increased, from day 8 onwards.

EXAMPLE 3

A preferred embodiment of a process in accordance with the invention forproducing an extract having appetite suppressant activity is illustratedschematically by way of example in FIGS. 3 and 4, which two Figurestogether illustrate the comprehensive process. However, various otherprocedures may be used, as will be understood by persons skilled in theart.

Referring to FIG. 3, plant material of the genus Trichocaulon or thegenus Hoodia is fed into a blender 3, eg a Waring blender, by way offeedline 1, with a solvent in the form of a methylene chloride/methanolsolution introduced via feedline 2. The homogenised product is fed vialine 4 into a separation stage 5, eg in the form of a filter orcentrifuge, and the residual plant material is removed via line 27.

The solvent/extract mixture is fed via line 6 into an evaporation stage7, where the solvent is removed, for example by means of a rotorevaporator. The dried crude extract is fed via line 8 into a furtherextraction stage 9 with the addition of a methylene chloride/watersolution introduced via feedline 29 for further extraction, and then toa separation stage 13 by way of line 11, where the water fraction isremoved via line 31. The dissolved extract fraction is fed via line 15into a drier stage 17 where the solvent is evaporated, for example by arotor evaporator.

Referring to FIG. 4, the dried extract is fed via line 10 into anextraction stage 12. A methanol/hexane solution is also fed via line 14into the extraction stage 12 for further purification and extraction ofthe dried extract. The extract/methanol/hexane mixture is fed via line16 into a separation stage 18, the hexane fraction is removed via line20, and the methanol/extract mixture, is then fed via line 22 into adrying stage 24. In the drying stage 24, the solvent is removed, eg byevaporation on a rotor evaporator.

The dried, partially purified active extract is fed via line 26 and withthe addition of methanol via line 28 into a solution stage 30, and thedissolved fraction is fed via line 36 to a chromatography column 38.

In the column 38 the methanol soluble fraction is further fractionated,using silica gel and a chloroform/30% methanol solvent, into differentfractions schematically indicated as fractions I to V. According to anactual fractionation procedure carried out by the Applicant, thefractionation procedure yielded the following fraction weights: I(3.9g); II(2.6 g); III(2.1 g); IV(1.1 g) and V(2.0 g). These fractions areindividually evaluated by a suitable bioassaying procedure (in a stepnot shown) and those fractions identified as fractions I and II,displaying marked appetite suppressant activity, are fed by feedlines 40and 42 into columns 44 and 46 respectively where they are furtherfractionated and purified by column chromatography, again by usingsilica gel and a 9:1 chloroform:methanol system.

The sub-fractions II(A)–(C) obtained from column 44 do not, whenassayed, display a noteworthy appetite suppressant activity, and may berecycled for further chromatography.

The sub-fractions I(A)–(L) obtained from column 46 are also evaluated(by an assaying step not shown), and the sub-fraction I(C) is found tohave marked appetite suppressant activity.

The sub-fraction I(C) is fed-via line 48 into column 50 for a furtherfractionation and purification, using silica gel and a 9:1 ethylacetatehexane eluent. Of the resultant purified fractions, fractionI(C)(ii) is found, after assaying, to possess marked appetitesuppressant activity.

The purified product is identified by nuclear magnetic resonancespectroscopy (as indicated in Tables 1 and 2 below), to be compound (1).

TABLE 1 ¹H (300.13 MHz) n.m.r. data for compound (1) CDCl₃ Compound (1)Hydrogen Atom J(HH)/Hz δ_(H)/p.p.m. Aglycone-3 — 3.522 m 6 — 5.381 m 1211.5, 4.1 4.607 dd 17 9.3, 9.3 3.157 dd 18 — 1.029 s 19 — 0.951 s 21 —2.164 s 3* 7.1, 1.5 6.888 qq 4* 7.1, 1.2 1.806 dq 5* 1.6, 1.2 1.853 dqCym-1′ 9.4, 2.1 4.816 dd 2′_(aq) 13.8, 3.7, 2.1 2.055 ddd 2′_(ax) 13.8,9.4, 2.6 1.552 ddd 3′ 3.7, 2.9, 2.6 3.776 ddd 4′ 9.4, 2.9 3.179 dd 5′6.3, 9.4 3.821 dd 6′ 6.3 1.279 d^(a) 3′-OMe — 3.408 s^(d) 1″ 9.4, 2.14.730 dd 2″ 13.8, 3.7, 2.1 2.108 ddd 2″^(aq) 13.8, 9.4, 2.6 1.601 ddd3″^(ax) 3.7, 2.9, 2.6 3.755 ddd 4″ 9.4, 2.9 3.239 dd 5″ 6.3, 9.4 3.898dd 6″ 6.3 1.243 d^(b) 3″-OMe — 3.392 s^(e) Thev-1″′ 7.7 4.273 d 2″′ 7.7,8.0 3.469 dd 3″′ 8.0, 2.9 3.099 dd 4″′ 9.3, 2.9 3.179 dd 5″′ 6.3, 9.33.351 dd 6″′ 6.3 1.183 d′^(c) 3″′-OMe — 3.622 s ^(a,b,c)in each columnmay be interchangeable. ^(d,e)in each column may be interchangeable,*Refers to the tigloate group atoms

TABLE 2 Relevant ¹³C (75.25 MHz) n.m.r. data for Compound (1) in CDCl₃Aglycone moiety Sugar moiety Carbon δ_(c)/p.p.m. Carbon δ_(c)/p.p.m. 137.04 T cym- 1′ 95.84 D 2 29.44 T 2′ 35.57 T 3 77.24 D 3′ 77.05 D 438.62 T 4′ 82.57 D 5 138.95 S 5′ 68.48 D 6 131.90 D 6′ 18.14 Q 7 27.30 T3′-OMe 57.93 Q 8 35.30 D 1″ 99.54 D 9 43.04 D 2″ 35.17 T 10  37.22 S 3″76.99 D 11  26.04 T 4″ 82.52 D 12  75.88 D 5″ 68.30 D 13  53.71 S 6″18.36 Q 14  85.69 S 3″-OMe 57.09 Q 15  34.36 T Thev- 1″′ 104.28 D 16 24.31 T 2″′ 74.62 D 17  57.18 D 3″′ 85.30 D 18  9.85 Q 4″′ 74.62 D 19 19.27 Q 5″′ 71.62 D 20  216.85 S 6″′ 17.75 Q 21  33.01 Q 3″′-OMe 60.60 Q 1* 167.60 S  2* 128.69 D  3* 137.66 D  4* 14.41 Q  5* 12.08 Q *Refersto the tigloate group atomsCompound (1)

IR data: 3440 cm⁻¹ (OH), 2910 cm⁻¹ (CH), 1700 cm⁻¹ (C=0) [α_(D)]²⁰₅₈₉=12.67° (C=3,CHCl₃) m.p. 147° C.–152° C.

Examples 4 to 13 illustrate the synthetic procedures whereby theintermediate compounds and steroid (15) may be prepared according to“the first alternative procedure”.

EXAMPLE 4 12β, 15α-Dihydroxy progesterone (17)

Cultures of Calonectria decora (ATCC 14767) are prepared by theinoculation of a culture medium comprised of sucrose (900 g), K₂HPO₄ (30g), Czapek concentrate (300 ml), corn steep liquor (300 ml) anddistilled water (30 l) (150×500 ml flasks). After 5 days of shaking at26° C., progesterone (16) (150 g) in a suspension of Tween 80 (0,1%soln., 1,5 l) is added to the flasks. The cultures are incubated for afurther 5 days and then worked-up by centrifugation, decantation,extraction of the medium with chloroform, and then evaporation to yieldthe dihydroxy. progesterone (17) (75 g, 45%)

¹H NMR (CDCl₃): 5.71 (1H, s, H-4); 4.12–4.22 (1H, m, H-15) 4.43 (1H, br,s, OH); 3.46–3.53 (1H, dd, J=4.6Hz, H-12); 2.16 Hz (3H, s, H-21); 1.18(3H, s, H-19); 0.74 (3H, s, H-18)

EXAMPLE 5 12β-Hydroxy-15α-(p-toluene sulfonyl)-progesterone (18)

The dihydroxy progesterone (17) (75 g, 0.22 mol) is dissolved in drypyridine (300 ml) and cooled to 0° C. p-Toluene sulfonyl chloride (46 g,0.24 mol) in dry pyridine (200 ml) is added dropwise to the reactionmixture at 0° C. The reaction is stirred overnight at 0° C., andquenched by the addition of H₂O (500 ml). The water layer is extractedwith ethyl acetate (1 l), and the organic extract washed withhydrochloric acid (6M, 3×1 l), aqueous saturated sodium bicarbonate (500ml), aqueous saturated sodium chloride (500 ml), and water (500 ml). Theorganic layer is dried (MgSO₄), filtered and evaporated to yieldp-toluene sulfonated progesterone (18) (98 g, 92%) as a viscous darkyellow oil.

¹H NMR (CDCl₃): 7.7 (2H, d, J=14 Hz, H-2.6); 7.34 (2H, d, J=8.4 Hz,H-3.5); 5.67 (1H, s, H-4); 4.86–4.93 (1H, m, H-15); 3.45–3.50 (1H, dd,J=4.6 Hz, H-12); 2.44 (3H, s, H-4Me); 2.15 (3H, s, H-21) 1.13 (3H, s,H-19); 0.74 (3H, s, H-18).

EXAMPLE 6 12β-Hydroxy-Δ¹⁴-progesterone (19)

A solution of the tosylated progesterone (18) (98 g, 0.19 mol) in2,4,6-trimethyl collidine (500 ml) is refluxed at 150° C. for 3 h. Thereaction mixture is cooled and poured into water (500 ml). The waterlayer is extracted with ethyl acetate (1 l), after which the organiclayer is washed with hydrochloric acid (6M, 3×1 l), aqueous saturatedsodium bicarbonate (500 ml), aqueous saturated sodium chloride (500 ml),and water (500 ml). After drying (MgSO₄) and filtering, the ethylacetate is evaporated and the crude mixture is purified by silica gelchromatography, eluting with acetone:

-   chloroform (1:10) to afford Δ¹⁴-progesterone (19) (50 g, 78%) as a    dark red oil.

¹H NMR (CDCl₃): 5.73 (1H, s, H-4), 5.28 (1H, dd, J=2.2 Hz, H-15), 4.41(1H, br, s, OH), 3.49–3.52 (1H, dd, J=4.3 Hz, H-12), 2.80–2.84 (1H, dd,J=9.2 Hz, H-17), 2.14 (3H, s, H-21), 1.19 (3H, s, H-19), 0.89 (3H, S,H-18).

EXAMPLE 7 3,12β-Diacetoxypregna-3,5,14-trien-20-one (20)

A solution of Δ¹⁴-progesterone (19) (50 g, 0.15 mol) in acetyl chloride(1.5 l) and acetic anhydride (750 ml) is refluxed for 2 hours. Thereaction mixture is poured into cold ethyl acetate (1 l) and aqueoussaturated sodium bicarbonate is added with stirring until theeffervescence ceases. The ethyl acetate layer is separated from thesodium bicarbonate layer and washed with further portions of aqueoussodium bicarbonate (3×700 ml), thereafter with aqueous saturated sodiumchloride (700 ml) and finally with water (700 ml). The organic layer isdried (MgSO₄), filtered and evaporated to afford the3,12β-diacetoxypregna-3,5,14-trien-20-one-(20) (60 g, 93%) as an orangeoil.

¹H NMR(CDCl₃): 5.68 (1H, s, H-4), 5.44 (1H, m, H-6), 5.31 (1H, dd, J=2.2Hz, H-15), 4.82–4.86 (1H, dd, J=4.5 Hz, H-12), 3.10–3.18 (1H, t, J=9.5Hz, H-17), 2.18 (3H, s, 3-Ac), 2.11 (3H, s, 12-Ac), 2.08 (3H, s, H-21),1.02 (3H, s, H-19), 1.01 (3H, s, H-18)

EXAMPLE 8 3,12β-Diacetoxy-20,20-ethylenedioxypregna-3,5,14-triene (21)

The diacetoxy compound (20) (60 g, 0.14 mol) is dissolved in benzene (1l) and ethylene glycol (60 ml) and p-toluene sulfonic acid (1 g) areadded. (The benzene is previously refluxed with a Dean-Stark trap). Themixture is refluxed with stirring and azeotropic removal of water for 16hours. Aqueous saturated sodium bicarbonate solution (500 ml) is addedto the cooled solution. This is then washed with brine (500 ml), andwith water (500 ml), and dried (MgSO₄). The solvent is evaporated andthe crude mixture purified by silica gel column chromatography, elutingwith ethyl acetate: hexane (2:8) to yield theethylenedioxypregna-3,5,14-triene (21) (35 g, 53%).

¹H NMR (CDCl₃): 5.68 (1H, s, H-4), 5.45 (1H, m, H-6), 5.31 (1H, dd,J=2.2 Hz, H-15), 4,73–4.85 (1H, dd, J=4.4 Hz, H-12), 3.78–3.98 (4H, m,ethylenedioxy), 2.16 (3H, s, 3-Ac), 2.04 (3H, s, 12-Ac), 1.29 (3H, s,H-21), 1.12 (3H, s, H-19), 1.02 (3H, s, H-18).

EXAMPLE 9 3β-12β-Dihydroxy-20,20-ethylenedioxypregna-5,14-diene-12acetate (22)

The dienolacetate (21) (35 g, 0,077 mol) is suspended in ethanol (500ml) and sodium borohydride (2.8 g, 0.074 mol) is added at 0° C. Themixture is allowed to warm to room temperature and stirred overnight.Most of the solvent is removed in vacuo and the mixture is diluted withwater (500 ml) and extracted with ethyl acetate (500 ml). Work-upfollowed by chromatography on silica gel with acetone/chloroform (1:10)yields the 3β-alcohol (22) (25 g, 80%).

¹H NMR (CDCl₃): 5.41 (1H, m, H-6), 5.28 (1H, dd, J=2.2 Hz, H-15),4.72–4.81 (1H, dd, J=4.4 Hz, H-12), 3.82–4.02 (4H, m, ethylene dioxy),3.45–3.59 (1H, m, H-3), 2.03 (3H, s, 12-Ac), 1.28 (3H, s, H-21), 1.10(3H, s, H-19), 1.01 (3H, s, H-18).

EXAMPLE 10 3β,12β-Dihydroxy-20,20-ethylenedioxypregn-5,14-diene (23)

The 3β-alcohol (22) (25 g, 60.2 mmol) in dry tetrahydrofuran (300 ml) isadded dropwise to a suspension of lithium aluminium hydride (2.7 g, 72.2mmol) in dry tetrahydrofuran (500 ml). The reaction mixture is stirredat room temperature for 24 hours after which water (2.7 ml) is carefullyadded and stirred for a further 10 min. Sodium hydroxide (15% soln, 2.7ml) is then added and the suspension stirred. After 10 min, water (8.1ml) is added and the suspension stirred for 10 minutes, filtered, dried(MgSO₄), and the solvent evaporated to afford the 3β,12βdihydroxypregna-diene (23) (20 g, 90%).

¹H NMR (CDCl₃): 5.36 (1H, m, H-6), 5.23 (1H, dd, J=2.2 Hz, H-15),3.94–4.06 (4H, m, ethylene dioxy), 3.41–3.52 (1H, m, H-3), 3.32–3.36(1H, dd, J=4.3 Hz, H-12), 1.31 (3H, s, H) 1.01 (3H, s, H-19), 0.96 (3H,s, H-18).

¹³C NMR (CDCl₃): 152,4 (c-14), 140.2 (c-5), 121.1 (c-15) 119.7 (c-6),111.1 (C-20), 79.8 (C-12), 71.6 (C-3), 63.7 and 63.6 (ethylene dioxy),58.8 (C-17), 19.0 (C-19), 11.9 (C-18).

3β,12β-Dihydroxy-14,15-epoxy-20,20-ethylenedioxypregn-5-ene;3β,12β-Dihydroxy-5,6-epoxy-20,20-ethylenedioxypregn-14-ene

N-Bromoacetamide (211 mg, 1.5 mmol) is added to a stirred solution ofthe 5,14-diene (23) (500 mg, 1.34 mmol) in acetone (100 ml), acetic acid(2.5 ml), and water (5 ml) at 0° C. After 15 min sodium sulphite (5%soln, 50 ml) is added to the reaction mixture. The acetone isevaporated, and the aqueous layer extracted with dichloromethane (3×50ml). The organic layer is dried (MgSO₄), filtered and evaporated.Pyridine (1 ml) is added to the product, and stirred for 0.5 h.Dichloromethane (100 ml) is then added to the reaction mixture, and thedichloromethane is washed with citric acid (5% soln, 3×100 ml),saturated sodium bicarbonate (50 ml), and water (50 ml). The organiclayer is dried (MgSO₄), filtered and evaporated to give the mixture of14,15- and 5,6-epoxides (360 mg, 69%) as a white foam. The mixture ofepoxides could not be separated by silica gel column chromatography.

EXAMPLE 11 3β,12β-Dihydroxy-14,15-epoxy-20,20-ethylenedioxypregn-5-ene(24)

The mixture of 14,15- and 5,6-epoxides (14.4 g, 37.0 mmol) in drytetrahydrofuran (200 ml) is added to a suspension of lithium aluminiumhydride (1.69 g, 44.4 mmol) in dry tetrahydrofuran (300 ml). Thereaction mixture is stirred at room temperature for 24 hours, afterwhich it is worked up as described earlier by the addition of water(1.69 ml), and sodium hydroxide (15% soln, 1.69 ml). After filtrationand evaporation of the solvent, the crude product is purified by silicagel column chromatography using methanol/chloroform (1:9) as solvent togive the unreacted 14.15 epoxy-20,20-ethylenedioxypregn-5-ene (24) (300mg, 2.1%).

¹H NMR (CDCl₃): 5.31 (1H, m, H-6), 3.82–3.98 (4H, m, ethylene dioxy),3.43–3.52 (1H, m, H-3), 3.41 (1H, s, H-15), 3.31–3.35 (1H, dd, J=4.3 Hz,H-12), 1.29 (3H, s, H-21), 1.17 (3H, s, H-19), 1.02 (3H, s, H-18)

¹³C NMR (CDCl₃): 139.8 (C-5), 120.8 (C-6), 112.1 (C-20), 77.2 (C-12),75.4 (C-14), 61.0 (C-15), 22.3 (C-21), 19.2 (C-19), 9.5 (C-18).

EXAMPLE 12 3β,12β,14β-Trihydroxy-20,20-ethylenedioxypregn-5-ene (25)

The 14,15-epoxide (24) (300 mg, 0.77 mmol) in dry tetrahydrofuran (10ml) is added to a suspension of lithium aluminium hydride (300 mg, 7.89mmol) in tetrahydrofuran and the reaction refluxed for 48 h. After theaddition of water (0.3 ml), sodium hydroxide (15% soln, 0.3 ml) andfiltration as described earlier, the mixture is purified by silica gelcolumn chromatography using methanol:chloroform (1:9) as solvent to givethe trihydroxy pregnene (25) (250 mg, 83%).

¹H NMR (CDCl₃):5.38 (1H, m, H-6), 3.98 (4H, m, ethylene dioxy),3.43–3.53 (1H, m, H-3), 3.25–3.32 (1H, dd, J=4.1 Hz, H-12), 1.32 (3H, s,H-21), 1.01 (3H, s, H-19), 0.98 (3H, s, H-18)

¹³C NMR CDCl₃): 139.1 (C-5), 122.1 (C-6), 112.2 (C-20), 85.1 (C-14),75.1 (C-12), 71.6 (C-3), 23.4 (C-21), 19.4 (C-19), 8.9 (C-18)

EXAMPLE 13 3β,12β,14β-Trihydroxy-pregn-5-ene (15)

The ethylenedioxypregnene (25) (250 mg, 0.64 mmol) is dissolved inacetic acid (13.4 ml) and water which after freeze drying affords thetrihydroxy steroid (15) (200 mg, 89%), m.p.: 228°–235° C. (lit.225°–235° C.), M+348, [α_(D)]²⁰ +35° (lit [α_(D)]²⁰+29°).

¹H NMR (CDCl₃): 5.39 (1H, m, H-6), 3.56–3.62 (1H, t, J=8.1 Hz, H-17),3.42–3.51 (1H, m, H-3), 3.28–3.39 (1H, dd, J=4,3 Hz, H-12), 2.23 (3H, s,H-21), 1.01 (3H, s, H-19), 0.90 (3H, s, H-18)

¹³C NMR (CDCl₃): 217.7 (C-20), 138.9 (C-5), 122.2 (C-6), 85.5 (C-14),73.6 (C-12), 71.6 (C-3), 57.0 (C-17), 55.1 (C-13), 43.6 (C-9), 42.1(C-4), 37.3 (C-1), 36.8 (C-10), 35.9 (C-8), 34.5 (C-15), 32.9 (C-21),31.5 (C-16), 30.1 (C-2), 27.4 (C-7), 24.4 (C-11), 19.4 (C-19), 8,3(C-18).

Examples 14 to 19 illustrate the synthetic procedures whereby theintermediate compounds and steroid (15) may be prepared according to“the second alternative procedure”.

EXAMPLE 14

20,20-Ethylenedioxy-3β-toluene-p-sulphonyloxy-pregn-5,14-diene-12β-olacetate (26)—A solution of p-toluenesulphonyl chloride (650 mg, 3.4mmol) in pyridine (10 ml) was added dropwise to a mixture of the20,20-Ethylenedioxypregna-5.14-diene-3β,12β-diol 12-acetate (22) (1.3 g,3.1 mmol) in pyridine (15 ml) at 0° C. The reaction mixture was leftstirring at room temperature for 24 hours after which water was added tothe reaction mixture. The solution was extracted with ethyl acetate(2×50 ml), the ethyl acetate layer was washed citric acid (5×50 ml),saturated sodium bicarbonate solution (100 ml), saturated sodiumchloride solution (100 ml) and water (100 ml). The ethyl acetate wasdried (MgSO₄), filtered, and evaporated and purified by flash columnchromatography using hexane-ethyl acetate (8:2 v/v) as the eluant togive the β-O-tosyl steroid (26), (1.5 g, 84%), as a yellow oil, (Found M570.271, C₃₂H₄₂O₇S requires: M 570.273).

δ_(H) 1.021 (3H, s, 19-H), 1.131 (3H, s, 18-H), 1.282 (3H, s, 21-H),2.021 (acetate0CH₃), 2.431 (3H, s, Ar-CH₃), 3.883 (4H, m, OCH₂CH₂O),4.750 (1H, dd, ³ J 10.8 Hz, 5.2 Hz, 12-H), 4.890 (1H, m, 30H), 5.281(1H, dd, ³ J 4.2 Hz, 2.1 Hz, 15-H), 5.388 (1H, m, 6-H), 7.341 (2H, d,³ J8.2 Hz, ArH), 7.746 (2H, d, ³ J 8.2 Hz, ArH).

δ_(c) 13.493Q (C-18), 19.002Q (C-19), 21.612Q (Ar-methyl)*, 21.671Q(C-21)*, 24.175Q (acetate methyl), 63.401T (ethylenedioxy), 63.498T(ethylenedioxy), 71.531S (C-13), 80.912D (C-12), 82.531D (C-3), 111.363S(C-20), 120.881D (C-15), 121.461D (C-6), 123.715–133.917 (Aromatic),139.903S (C-14), 151.722S (C-5) 170.819S (ester carbonyl). * may beinterchanged

EXAMPLE 15

-   20,20-Ethylenedioxy-3α,5-cyclo-5α-pregn-14-ene-6β,12β-diol-12-acetate    (27)—A solution of 3β-toluene-p-sulphonyloxy-pregn-5, 14-diene (26)    (1.2 g, 2.1 mmol) and potassium acetate (2.2 g, 22.4 mmol) in water    (250 ml) and acetone (500 ml) was refluxed at 60° C. for 16 hours.    The acetone was evaporated and the water was extracted with ethyl    acetate (200 ml). The ethyl acetate was dried (MgSO₄), filtered, and    evaporated. Flash chromatographic separation of the mixture using    chloroform-acetone (9:1 v/v) as the eluant gave the 3α,5-cyclo    derivative (27), (530 mg, 610%) as a yellow oil, (Found M 416.262,    C₂₅H₃₆O₅ requires: M 416.263).

δ_(H) 0.288 (1H, dd, ³ J 8.1 Hz, 4.9 Hz, 4-H_(a)) 0.477 (1H, dd, ³ J 4.4Hz, 4.4 Hz, 4-H_(b)), 1.025 (3H, S, 19-H), 1.121 (3H, s, 18-H), 1.256(3H, s, 21-H), 1.989 (3H, s, acetate-CH₃), 3.302 (1H, dd, ₃ J 2.8 Hz 2.8Hz, 6-H), 3.784–3.947 (4H, m, OCH₂CH₂O), 4.721 (1H, dd, ³ J 8.5 Hz, 5.6Hz, 12-H), 5.232 (1H, dd, ³ J 3.9 Hz, 1.9 Hz, 15-H).

δ_(c) 11.678T(C-4), 12.298Q(C-18), 19.971Q (C-19), 23.623Q(C-21),24.153Q (acetate methyl), 63.700T (ethylenedioxy), 63.788T(ethylenedioxy), 73.591D (C-6), 80.551D (C-12), 111.126S (C-20),118.778D (C-15), 152.959S (C-14), 170.991S (ester carbonyl).

EXAMPLE 16

-   20,20-Ethylenedioxy-3α,5-cyclo-5α-pregn-14-ene-6β,12β-diol (28)—A    solution of the 3α, 5-cyclo derivative (27), (500 mg, 1.2 mmol) in    tetrahydrofuran (20 ml) was added dropwise to a suspension of    lithium aluminium hydride (50 mg, 1.3 mmol) in tetrahydrofuran (10    ml). The reaction mixture was stirred for 4 hours and quenched by    the addition of water (50 μl). After 30 minutes, sodium hydroxide    was added (15% solution, 50 μl) and stirring continued for a further    30 minutes. Water (150 μl was added and the reaction mixture was    filtered. The tetrahydrofuran was dried (MgSO₄) filtered and    evaporated and flash chromatographic purification using    chloroform-acetone (8:2 v/v) as the eluant to give the diol (28),    (370 mg, 83%) as an oil, (Found M 374.250, C₂₃H₃₄O₄ requires: M    374.252)    -   δ_(H) 0.298 (1H, dd, ³ J 8.1 Hz, 4.9 Hz, 4-H₂), 0.510 (1H, dd, ³        J 4.4 Hz, 4.4 Hz, 4-H_(b)), 0.985 (3H, s, 19-H), 1.055 (3H, s,        18-H), 1.325 (3H, s, 21-H), 3.318 (1H, dd, ³ J 3.0 Hz, 3.0 Hz,        6-H),), 3.363 (1H, dd, ³ J 11.4 Hz, 4.2 Hz, 12-H), 4.019 (4H, m,        OCH₂Ch₂O) 4.622 (1H, s, OH), 5.255 (1H, dd, ³ J 3.9 Hz, 1.9 Hz,        15-H)    -   δ_(c) 11.681T(C-4), 12.243Q(C-18), 19.844Q (C-19),        23.604Q(C-21), 63.620T (ethylenedioxy), 63.733T (ethylenedioxy),        73.569D (C-6), 77.478D (C-12), 111.125S (C-20), 118.702D (C-15),        152.912S (C-14).

EXAMPLE 17

-   20,20-Ethylenedioxy-14,15β-epoxy-3α,    5-cyclo-5α,14β-pregnane-6δ,12β-diol (29) -N-bromoacetamide (150 mg,    1.1 mmol) was added to a solution of the    20,20-ethylenedioxy-3α,5-cyclo-5α-pregn-14-ene-6β, 12β-diol (28)    (340 mg, 0.91 mmol) in acetone (20 ml), water (0.25 ml) and acetic    acid (0.25 ml) at 0° C. After 15 min., sodium sulphite (5% solution,    20 ml) was added to the reaction mixture. The acetone was evaporated    under reduced pressure and the remaining solution was extracted with    dichloromethane (3×30 ml). The dichloromethane layer was dried    (MgSO₄), filtered and evaporated to a concentrated volume (50 ml).    Pyridine (0.5 ml) was added to the mixture and stirred for a further    1 hour after which the dichloromethane layer was washed with a    citric acid solution (5%, 3×30 ml), saturated sodium bicarbonate    solution (30 ml) and water (30 ml). The dichloromethane layer was    dried (MgSO)₄), filtered and evaporated and purified by flash column    chromatography using chloroform-methanol (9.5:0.5 v/v) as the eluant    to give the epoxide (29) (180 mg, 51% as a foam, (Found M 390.245,    C₂₃H₃₄O₂ requires: M 390.247).

δ_(H) 0.287 (1H, dd, ³ J 8.1 Hz, 4.9 Hz, 4-H_(a)), 0.501 (1H, dd, ³ J4.4 Hz, 4.4 Hz,4-H_(b)), 0.978 (3H, s, 19-H), 1.048 (3H, s, 18-H), 1.321(3H, s, 21-H), 3.318 (1H, dd, ₃ J 3.1 Hz, 3.1 Hz, 6-H), ), 3.355 (1H,dd, 3 J 11.2 Hz, 4.1 Hz, 12-H), 3.491 (1H, s, 15-H), 4.001 (4H, m,OCH₂Ch₂O), 4.901 (1H, s, OH).

δ_(c) 11.668T(C-4), 11.973Q(C-18), 19.515Q (C-19), 23.519Q(C-21),59.910D (C-15), 63.601T (ethylenedioxy), 63.713T (ethylenedioxy),72.501S (C-14), 73.571D (C-6), 77.471D (C-12), 111.085S (C-20).

EXAMPLE 18

20,20-Ethylenedioxy-6β, 12β, 14-trihydroxy-3α, 5-cyclo-5α, 14β-pregnane(30)—A solution of the epoxide (29) (170 mg, 0.44 mmol) intetrahydrofuran (10 ml) was added to a suspension of lithium aluminiumhydride (20 mg, 0.53 mmol) in tetrahydrofuran (5 ml). The reactionmixture was refluxed for 2 hours after which water (20 μl) was added andstirring continued for 05 hour. Sodium hydroxide solution (15%, 20 μl)was added and stirring continued for a further 0.5 hour. A furtherquantity of water was added (60 μl) and the suspension was stirred for 1hour. After filtration, the suspension was dried (MgSO₄) filtered, andthe tetrahydrofuran was evaporated. Flash chromatographic separation ofthe resulting mixture eluting with chloroform-methanol (9:1 v/v) gavethe required triol (30), (90 mg, 53%) as a clear oil, (Found M 392.261,C₂₃H₃₈O₅ requires: M 392.263).

δ_(H) 0.287 (1H, dd, ³ J 8.1 Hz, 4.9 Hz, 4-H₂), 0.510 (1H, dd, ³ J 4.4Hz, 4.4 Hz, 4-H_(b)), 0.971 (3H, s, 19-H), 1.042 (3H, s, 18-H), 1.319(3H, s, 21-H), 3.321 (1H, dd, ³ J 3.0 Hz, 3.0 Hz, 6-H), 3.321 (1H, dd, ³J 11.1 Hz, 3.9 Hz, 12-H), 3.561 (1H, s, OH), 4.084 (4h, m, OCH₂Ch₂O)4.671 (1H, s, OH)

δ_(c) 11.668T(C-4), 11.971Q(C-18), 19.511Q (C-19), 23.520Q (C-21),63.612T (ethylenedioxy), 63.711T (ethylenedioxy), 73.483D (C-6), 76.051D(C-12), 84.307S (C-14), 111.099S (C-20).

EXAMPLE 19

3β, 12β, 14-Trihydroxy-14β-pregn-5-en-20-one (15)—A mixture of the triol(30) (80 mg, 0.20 mmol) in acetone (20 ml) and hydrochloric acid (1M, 10ml) was refluxed at 60° C. for 2 hours. The reaction mixture was cooledand saturated sodium bicarbonate solution *(20 ml) was added. Theacetone was evaporated and the aqueous layer extracted with chloroform(3×20 ml), the chloroform layer was dried (MgSO₄), filtered andevaporated to give the epimeric trihydroxy steroids (15a, 15b) (42 mg,61%). Separation of the epimeric mixture (15a, 15b) (15 mg) was achievedby flash chromatographic separation using chloroform methanol (9:1 v/v)as the eluant to give the pure 17β-epimer (15a), (10 mg), m.p. 224–229°C. (acetone), (lit. 226–223°), (Found M 348.234,C; 72.32, H 9.21%C₂₁H₃₂O₄ requires: C, 72.38; H 9.26%, M 348.236), and the 17α-epimer(15B) (3 mg), m.p. 183–191° C. (acetone), (lit 184–196°).

3β, 12β, 14-Trihydroxy-14β-pregn-5-en-20-one (15a): δ_(H) 0.963 (1H, s,19-H), 1.192 (3H, s, 18-H), 2.236 (3H, s 21-H), 3.325 (1H, dd, ³ J 11.2Hz, 3.9 Hz, 12-H), 3.464 (1H, s, OH), 3.5140 (1H, m, 3-H), 3.598 (1H,dd, ³ J 9.6 Hz, 9.6 Hz, 17-H), 4.255 (1H, s, OH), 5.383 (1H, m, 5-H)

δ_(c) 8.275Q (C-18), 19.414Q (C-19), 24.400T (C-11) 24.581T (C-16),27.443T (C-7), 30.062T (C-2), 32.972Q (C-21), 34.543T (C-15), 35.864D(C-8), 36.975S (C-10), 37.337T (C-1), 42.144T (C-4), 43.565D (C-9),55.101S (C-13), 57.038D (C-17), 71.597D (C-3), 73.558D (C-12), 85.566S(C-14), 122.223D (C-6), 138.932S (C-5), 217.011S (C-20).

3β, 12β, 14-Trihydroxy-14β-pregn-5-en-20-one (15b): δ_(H) 0.996 (1H, s,19-H), 1.144 (3H, s, 18-H), 2.221 (3H, s 21-H), 3.339 (1H, dd, ³ J 9.4Hz, 9.4 Hz, 17-H), 3.492 (1H, m, 3-H), 3.629 (1H, dd, ³ J11.1 Hz, 3.9Hz, 12-H), 3.712 (1H, s, OH), 4.325 (1H, s, OH), 5.383 (1H, m, 5-H).

Examples 20 to 28 illustrate the procedures whereby the intermediatecompounds may be prepared to form the first. monosaccharide (40).

EXAMPLE 20 Methyl-4, 6-0-benzylidene-α-D-glucopyranoside (32)

A mixture of methyl-α-D-glucopyranoside (30 g, 0,15 mol), benzaldehyde(70 ml) and zinc chloride (20 g) is stirred at room temperature for 24hours. The reaction product is poured into ice water and stirringcontinued for 15 min. The white precipitate is filtered and washed withdiethyl ether. The solid material is stirred with a solution of sodiummetabisulphite (10% soln), for 15 min, filtered and washed with water.The solid material is crystallized from chloroform and ether to yieldthe benzylidene product (32) (31 g, 72%).

EXAMPLE 21 Methyl-4,6-0-benzylidene-2-0-tosyl-α-D-glucopyranoside(33)

p-Toluene sulfonyl chloride (25 g, 1.2 eq) in pyridine (100 ml) is addeddropwise to a solution of the benzylidene glucose (32) (31 g, 0.12 mol)in pyridine (100 ml) at 0° C. The reaction is stirred at roomtemperature for 48 hours. Ice is added to the reaction mixture. Theresulting white solid material is washed with water and recrystallizedfrom hot ethanol to yield the tosylated glucose (33) (28 g, 60%).

EXAMPLE 22 Methyl-4,6-0-benzylidene-3-0-methyl-α-D-altropyranoside (34)

The tosylate (33) (28 g, 64 mmol) in a solution of is sodium (7 g) inmethanol (150 ml) is heated at 110° C. for 48 hour in an autoclave. Thereaction vessel is cooled and solid carbon dioxide is added to thereaction mixture. After filtration, the methanol is evaporated and thesolid material is then taken up in water. The aqueous layer is extractedwith chloroform (×3). The chloroform is dried (MgSO₄), filtered andevaporated. The crude mixture is purified by silica gel columnchromatography eluting with chloroform : acetone (9:1) to yield thealtroside (34) (10 g, 52% )

EXAMPLE 23Methyl-6-bromo-4-0-benzoyl-3-0-methyl-6-deoxy-α-D-altropyranoside (35)

The benzylidene altroside (34) (10 g, 33 mmol) is added to a solution ofN-bromosuccinimide (7.6 g) and barium carbonate (20 g) in carbontetrachloride and the reaction mixture is refluxed at 75° C. for 3hours. The reaction mixture is filtered and the carbon tetrachloridelayer is washed with water. The organic layer is dried (MgSO4), filteredand evaporated to yield 6-bromo-altroside (35), (9 g, 69%).

EXAMPLE 24 Methyl-4-0-benzoyl-3-0-methyl-6-deoxy-α-D-altropyranoside(36)

Sodium borohydride (18 g) in water (30 ml) is added dropwise to asolution of the bromoaltroside (35) (9 g, 23 mmol) and nickel chloride(18 g) in ethanol (300 ml) at 0° C. The reaction mixture is refluxed at75° C. for 1 hour and then it is filtered. The ethanol is evaporated andthe remaining aqueous layer is extracted with chloroform (×3). Thechloroform is dried (MgSO₄), filtered and evaporated, to yield the6-deoxy-altroside (3.6) (5 g, 72%).

EXAMPLE 25 4-0-Benzoyl-3-0-methyl-6-deoxy-αβ-D-phenylthioaltropyranoside(37)

Phenylthiotrimethylsilane (5 ml) and trimethylsilyltrifluoromethanesulphonate (2 ml) are added at 0° C. to a solution of the6-deoxy-altroside (36) (5 g, 17 mmol) in dichloromethane (200 ml). Thereaction mixture is stirred at room temperature for 6 hours. Saturatedsodium bicarbonate is added to the reaction mixture. The dichloromethanelayer is dried (MgSO₄), filtered and evaporated. The crude mixture ispurified by silica gel column chromatography eluting withchloroform:acetone (9:1) to yield the αβ-phenylthioaltroside (37) (4 g,63%).

EXAMPLE 264-0-Benzoyl-3-0-methyl-2-phenylthio-2,6-dideoxy-αβ-D-fluorocymaropyranoside(38)

Diethylaminosulphurtrifluoride (0.65 g) is added rapidly to a solutionof the αβ-phenylthioaltroside (37) (0.5 g, 1,33 mmol) in dichloromethaneat 0° C. The reaction is stirred for 0,5 h at 0° C. and then saturatedsodium bicarbonate is added. The dichloromethane is separated from theaqueous layer, dried (MgSO₄), filtered and evaporated to yield theαβ-fluorocymarose (38) (450 mg, 90%).

EXAMPLE 274-0-Benzoyl-3-0-methyl-2-0-t-butyldimethylsilyl-αβ-D-phenylthio-altroside(39)

The 6-deoxy altroside (37) (5 g) is silylated usingt-butyldimethylsilylchloride (3 g) and imidazole (3 g) in pyridine (50ml). The reaction is worked-up by extracting with ethyl acetate, washingthe ethyl acetate with hydrochloric acid (6 N), then with sodiumbicarbonate, and finally with water. The ethyl acetate layer is dried(MgSO₄), filtered and evaporated to yield the silylated benzoylphenylthioaltroside (39) (80%).

EXAMPLE 28 3-0-methyl-2-0-t-butyldimethylsilyl-αβ-D-phenylthioaltroside(40)

The silylated benzoyl phenylthioaltroside (39) (6 g) is treated withsodium methoxide (100 ml) for 4 hours. The methanol is evaporated andwater is added to the reaction. The water layer is acidified (pH 5,ACOH) and extracted with ethyl acetate. The ethyl acetate is washed withwater, dried (MgSO₄), filtered and evaporated to yield silylated methylphenylthioaltroside (40) (75%).

Examples 29 to 37 illustrate the procedures synthetic whereby theintermediate compounds may be prepared to form the second monosaccharide(50).

EXAMPLE 29 1.2:5.6-Di-0-isopropylidene-α-D-glucofuranose (42)

Sulfuric acid (40 ml) is added dropwise to a solution of α-D-glucose(41) (50 g. 0.28 mol) in acetone (1 l) at 0° C. The reaction mixture isstirred for 24 h and then it is neutralized using sodium hydroxide (6M). The acetone is evaporated and the aqueous layer is extracted withchloroform (×2). The chloroform is dried (MgSO₄) filtered andevaporated. Crystallization from cyclohexane yielded thedi-isopropylidene glucose (42) (41 g. 57%).

EXAMPLE 30 1.2:5.6-Di-0-isopropylidene-3-0-methyl-α-D-glucofuranose (43)

The α-D-glucofuranose (42) (41 g, 0.16 mol) in tetrahydrofuran (300 ml)is added dropwise to a suspension of sodium hydride (5 g) intetrahydrofuran (200 ml). After 0.5 h, methyl iodide (25 g) intetrahydrofuran (100 ml) is added dropwise to the reaction mixture whichis then stirred for 24 h. Water is added to the reaction mixture whichis then extracted with ether (×3). The ether layer is dried (MgSO₄),filtered and evaporated to yield the methyl protected glucose (43) (38g. 83%).

EXAMPLE 31 3-0-Methyl-αβ-D-glucopyranoside (44)

The methyl diisopropylidene compound (43) (38 g. 0.14 mol) is dissolvedin acetic acid (50%, 700 ml) and the solution refluxed for 18 h. Aftercooling the acetic acid is evaporated. The crude product is purified bycolumn chromatography eluting with chloroform:methanol:acetone:water(70:27:2:1) to yield 3-0-methyl-αβ-glucopyranoside (44) (13 g. 50%).

EXAMPLE 32 Methyl 3-0-methyl-αβ-D-glucopyranoside (45)

The 3-0-methyl-αβ-glucopyranoside (44) (10 g) is dissolved in methanol(50 ml) and HCl (conc.) (1 ml) and refluxed overnight. Solid NaHCO₃ isadded and the reaction is filtered. The methanol is evaporated to give1,3-di-0-methyl-αβ-D-glucopyranoside (45), (95%).

EXAMPLE 33 Methyl4,6-0-benzylidene-3-0-methyl-αβ-glucopyranoside (46)

The glucopyranoside (45) (8 g) is stirred at room temperature in asolution of benzalaldehyde (20 ml) and zinc chloride (5 g). After 24hours, ice is added and the aqueous layer is extracted with chloroform.The chloroform layer is dried (MgSO₄), filtered and evaporated. Thebenzalaldehyde is removed by vacuum distillation and the product ispurified by silica gel column chromatography eluting withacetone:chloroform (0.5:9.5), to yield benzylidene-αβ-glucopyranoside(46) (60%).

EXAMPLE 34 Methyl4-0-benzoyl-0-methyl-6-deoxy-αβ-glucopyranoside (47)

The benzylidene compound (46) (5 g) is refluxed at 80° C. in a mixtureof N-bromosuccinimide (3.7 g) and barium carbonate (4 g) in carbontetrachloride. After 4 hours, the reaction is filtered and the carbontetrachloride is washed with water, dried (MgSO₄), filtered andevaporated to give the bromo compound (70%).

The bromo compound (4.3 g) is dissolved in a solution of ethanol (300ml) and nickel chloride (8.6 g) at 0° C. To this solution, sodiumborohydride (8.6 g) in water (50 ml) is added dropwise over a period of15 minutes. The reaction mixture is refluxed at 100° C. for 45 minutes,cooled, filtered and evaporated. Chloroform is added, and the chloroformlayer is washed with water, dried (MgSO₄), filtered and evaporated togive the 6-deoxy sugar (47) (70%).

EXAMPLE 354-0-Benzoyl-3-0-methyl-1-phenylthio-6-deoxy-αβ-glucopyranoside (48)

The 6-deoxy glucopyranoside (47) (3 g) is dissolved in dichloromethane(50 ml). To this solution, phenylthiotrimethylsilane (2 g) andtrimethylsilyltrifluoromethanesulphonate (0.2 ml) are added. Thesolution is stirred at room temperature overnight, after which saturatedsodium bicarbonate is added. The dichloromethane layer is dried (MgSO₄),filtered and evaporated. The product is purified by silica gel columnchromatography eluting with ethyl acetate:hexane (2:8), to give thecompound (48) (60%).

EXAMPLE 364-0-Benzoyl-3-0-methyl-2-0-pivaloyl-1-phenylthio-6-deoxy-αβ-glucopyranoside(49)

To a solution of the glucopyranoside (48) (2 g) in pyridine (20 ml),pivaloyl chloride (2 ml) is added. The solution is stirred at roomtemperature overnight after which water is added. The aqueous layer isextracted with ethyl acetate, and the organic layer is washed with HCl(6 N). The organic layer is dried (MgSO₄), filtered and evaporated togive the pivaloyl ester (49) (80%).

EXAMPLE 374-0-Benzoyl-3-0-methyl-2-0-pivaloyl-1-fluoro-6-deoxy-β-glucopyranoside(50)

N-Bromosuccinimide (1.2 g) and diethylaminosulphur trifluoride (1.2 g)are added to a solution of the pivaloyl ester (49) (2 g) indichloromethane (100 ml) at 0° C. After 1 hour, saturated sodiumbicarbonate is added. The dichloromethane layer is dried (MgSO₄),filtered and evaporated. The β-fluoropyranoside (50) is purified bysilica gel column chromatography eluting with ethyl acetate:hexane(2:8), (yield 45%).

Example 38 illustrates the synthetic procedure whereby the compound3-0-(4-0-benzoyl-2-phenylthio-β-D cymaropyranosyl)-12,14β-dihydroxy-pregnan-5-ene-20-one(51) may be prepared.

EXAMPLE 38 3-0- [4-0-benzoyl-2-phenylthio-β-D-cymaropyranosyl]-12,14β-dihydroxy-pregn-5-en-20-one (51)

Tin chloride (190 mg, 1 mmol) is added to a solution of 3, 12, 14β-trihydroxy pregnan-5-ene-20-one (15) (100 mg, 0.28 mmol) and thefluorocymaropyranoside (38) (210 mg, 0. 56 mmol), in dry diethyl etherand 4 Å molecular sieves at −15° C. The reaction mixture is maintainedat −15° C. for 3 days. Saturated sodium bicarbonate is added to thereaction mixture. The ether layer is dried (MgSO₄), filtered andevaporated. The product is purified by silica gel column chromatographyeluting with chloroform methanol (9, 5:0, 5) to yield the glycoside (51)(30 mg. 15 %).

Examples 39 to 41 illustrate the synthetic procedures whereby thecymarose and thevetose moieties may be coupled.

EXAMPLE 39 Thevetose-cymarose dissaccharide (53)

A solution of thevetose (50 A) (1.5 g), cymarose (40) (1.3 g), andmolecular sieves 4Å in dichloromethane is stirred at room temperaturefor 1 hour. The reaction mixture is cooled to −15° C., and tin (II)chloride (0.8 g) and silver trifluoromethanesulphonate (1.1 g) areadded. The mixture is stirred at −15° C. for 16 hours, after whichtriethylamine (0.5 ml) is added. The reaction product is filtered andthe dichloromethane is evaporated. The dissaccharide (53) is purified bysilica gel column chromatography eluting with ethyl acetate:hexane(2:8), yield 15%.

EXAMPLE 40 Thevetose-cymarose dissaccharide (54)

To a solution of the dissaccharide (53) (200 mg) in tetrahydrofuran (20ml), tetrabutylammonium fluoride (0.4 ml) is added. The mixture isstirred at room temperature for 1 hour, after which saturated sodiumbicarbonate is added. The reaction mixture is extracted with ethylacetate and the ethyl acetate layer is dried (MgSO₄), filtered andevaporated. The dissaccharide (54) is purified by silica gel columnchromatography (acetone:chloroform, 0.5:9.5) yield 60%.

EXAMPLE 41 Thevetose-Cymarose Dissaccharide (55)

To a solution of the dissaccharide (54) (80 mg) in dichloromethane (10ml), diethylamino sulphur trifluoride (80 μl) is added at 0° C. Afterstirring at 0° C. for 0.5 hour, saturated sodium bicarbonate and moredichloromethane are added. The dichloromethane is dried (MgSO₄),filtered and evaporated. Purification by silica gel columnchromatography (ethyl acetate:hexane 1:9), gives the dissaccharide (55)in a 65% yield.

EXAMPLE 42

The results of the following three bioassays on the appetite suppressantare set out below, viz.

a) Irwin Test;

b) Acute Toxicity Test; and

c) Oral Dose Anorectic Test.

a) Irwin Test

The purpose of this test was to evaluate the appetite suppressant of theinvention produced from a plant extract as hereinbefore described,according to the reduced animal Irwin test for tranquillising andsedative action.

Experimental Procedure

The appetite suppressant was extracted from plant material by theApplicant by the method as hereinbefore described and administered totwo of four groups of three animals each: one group receiving notreatment, one group receiving the solvent dimethylsulfoxide (DMSO), onegroup receiving the test sample at 50 mg/kg; and one group receiving thetest sample at 300 mg/kg. Treatment took place by intraperitonealinjection, and observations were made at specific intervals up to fivehours post treatment. Only symptoms other than those observed in theDMSO-treated animals were used in the interpretation of the results.

Results

It was clear that the solvent, DMSO, had a marked effect on the animals,especially on the heat regulating mechanism. Body temperatures of allthe animals treated with the solvent, alone or together with the testsample, showed a marked drop.

Animals in the low dose group showed decreased dispersion in the cageand decreased locomotor activity, as in all the other groups, includingthe control group. Apathy was seen in the same degree as in theDMSO-treated group. Decreased respiration was observed 15–60 minutesafter treatment. Ptosis (closing of the eyelids) was also observed to alarger degree than in the DMSO group. A pinna (ear) response was seen aswell as a positive finger response, indicating fearfulness. Bodytemperature dropped to 32,7° C. after treatment.

Animals in the high dose group showed as in the other groups an initialdecreased dispersion in the cage and decreased locomotor activity, butshowed increased dispersion and locomotor activity before death, whichoccurred approximately 1 hour after treatment. Severe clonic symmetricalconvulsions occurred 30 minutes after treatment. Respiration decreasedinitially, but increased before death. A pinna (ear) response wasdelayed and a positive finger response was observed, indicatingfearfulness, both as observed in animals in the low dose group. Bodytemperature dropped to 30,7° C. after treatment. Increased positionalpassivity was observed as well as decreased body tone. Abnormal limbrotation was observed, the grip strength decreased, no pain response waspresent and loss of righting reflex occurred.

Discussion

When compared with the control and DMSO-treated animals, animalsreceiving the low dose (50 mg/kg) only showed decreased respiration andan increased degree of ptosis. Animals receiving the high dose (300mg/kg) of the test sample reacted very intensely by showing convulsionsand death. All other observations made in these animals can be ascribedto the animals being in convulsions and dying. Signs suggestive oftranquillising and sedative actions such as marked decreased dispersionin the cages, decreased locomotor activity and apathy in the test groupsthat could be ascribed to the test sample were not seen.

It can therefore be concluded that the test sample is lethal to mice at300 mg/kg and has respiratory suppressive effects on mice at 50 mg/kg,when given intraperitoneally with DMSO as solvent.

b) Acute Toxicity Test

The purpose of this test was to gain information on the toxicity of thetest sample.

Experimental Procedure

A plant extract prepared in accordance with the invention ashereinbefore described, and having appetite suppressive action waspurified and one test sample was tested at increasing doses by oraltreatment in mice. Two animals were used per dose group, except in thehighest dose group where only one animal was treated. Animals wereexamined for good health and their body masses determined on the day oftreatment.

Doses ranged from 100 mg/kg up to 3 028.5 mg/kg. The dose was calculatedand mixed into prepared potato starch, so that each animal received atotal dose of 0.2 ml. Animal 13 received 0.25 ml. Potato starch wasprepared by mixing 20 g starch into a small volume of cold water, andadding it to boiling water, to make up a volume of 1 litre. Thesuspension was allowed to cool to room temperature before dosing.

Animals in groups 1 and 2 were treated on the same day. They wereobserved for 24 hours and if no signs of toxicity developed, the nextgroup was treated. The same approach was followed until all the animalswere treated. This schedule was followed to ensure that animals were notunnecessarily treated when an acute toxic dose had been reached in theprevious group.

Animals were observed for clinical signs of toxicity immediately (1–2hours) after treatment and daily thereafter. Body mass was determinedonce a week and total food and water intakes of each animal weremeasured.

Surviving animals were euthanased by intraperitoneal injection ofpentobarbitone sodium (commercially available under the trade nameEuthanaze, Centaur^(R)) on day 14 of the experiment. A post-mortemexamination was performed on these animals, as well as on the one animalwhich died during the experiment. Samples for histopathology werecollected.

Results

Group 1 (Control Group)

No clinical signs of toxicity were observed during the 14-dayobservation period. Food and water intakes were within the normalparameters. Changes in body mass were also within normal parameters. Nohistopathological changes were recorded in the liver samples.

Group 2 (100 mg/kg)

No clinical signs of toxicity were observed during the observationperiod. Food and water intakes were normal and changes in body mass overthe observation period were also normal. No macroscopical pathology wasobserved and no histopathological or morphological changes were recordedin the liver samples.

Group 3 (200 mg/kg)

Animals in this group showed no clinical symptoms of toxicity during theexperiment. Food and water intakes were normal, as was the change inbody mass. No macroscopic pathology was observed, but the livers showedhistopathological changes on examination. Cloudy swelling of thehepatocytes was mild in animal 6, but moderate in animal 5. Moderatehydropic degeneration also occurred in the hepatocytes of animal 5.

Group 4 (400 mg/kg)

No clinical signs of toxicity were observed during the observationperiod, and no macroscopic pathology was observed during the post-mortemexamination. Moderate cloudy swelling and mild hydropic changes of thehepatocytes were observed on histology.

Water and food intakes and the increase in body mass in animal 7 werenormal. Animal 8 consumed almost double the total food intake of animal7 (144.6 g and 73.9 g respectively), but the increase in body mass wasonly 0.81 g compared to 2.7 g.

Group 5 (800 mg/kg)

One animal (animal 10) died three hours after dosing without showing anyspecific signs. The other animal (animal 9) survived the entireobservation period without any signs of toxicity. Water intake in thesurviving animal was normal (42.42 ml), while food intake was high(134.2 g). The body mass increased by 2.85 g which was the highest ofall animals in the experiment.

At the post-mortem examination of animal 10, which died shortly afteroral dosing, the lungs were congested. No foreign body reaction whichwould have indicated inhalation of test material was present. Nomacroscopic pathology was observed in animal 9. Mild cytoplasmicvacuolisation (hydropic degeneration) was present in animal 10, butmoderate in animal 9. The glandular cytoplasmic appearance of the liverwas classified as moderate in both animals.

Group 6 (1 600 mg/kg)

None of the animals presented any clinical signs of toxicity during theduration of the experiment. No macroscopic pathology was observed atpost-mortem examination, but moderate degenerative changes in the liverof animal 11 were observed at histopathological examination. Animal 12showed moderate cloudy swelling and mild hydropic changes of thehepatocytes. Food and water intakes were normal, as was the increase inbody mass over the experimental period.

Group 7 (3 028.5 mg/kg)

Only one animal was treated at this dose. This animal showed no signs oftoxicity during the observation period, and no macroscopic pathology wasobserved. At histopathological examination, moderate cloudy swelling andhydropic degeneration of the hepatocytes was observed. The animal showeda loss of body mass over the observation period (−0.82 g), but food andwater intakes were normal.

Discussion

Since a very small number of animals were used in each dose group, it isdifficult to make any conclusions. The fact that only one animal died ata low dose rate, without showing any symptoms, might indicate that deathwas not related to the test sample, but due to stress during and/orafter treatment. No animals in higher dose groups died or showed anysigns of toxicity, which further supports this assumption.

The increased food intake observed in animal 8 could possibly beascribed to excessive spillage of food as was reflected in the smallincrease in body mass. It should be kept in mind that all the animals inthis experiment were only treated once, and that it is unlikely that anappetite suppressor will have a marked influence on either the food orwater intakes, or body mass over a 14 day period, as was the case inthis experiment.

From the histopathological examination of the liver samples, it wasclear that the pathological changes were dose related, with animalsreceiving higher doses showing the extensive changes. The pathologyobserved was not metabolic of nature, but possibly test sample-induced.The changes were only degenerative and therefore reversible. No signs ofirreversible hepatocellular changes were observed.

It can, therefore, be concluded that only one animal died at a lowerdose (800 mg/kg), but that the death was possibly not test samplerelated. None of the other animals in any of the dose groups showed anysigns of toxicity during the 14 day observation period after treatment,or died as result of the treatment. A single oral dose of the testsample induced reversible dose-related hepatocellular changes.

c) Oral Dose Anorectic Test

The purpose of this test was to determine the activity of a plantextract prepared in accordance with the invention, and the minimumeffective dose, and at the same time investigate any possibleside-effects such as respiratory suppression, as experienced in theIrwin Test (referred to above).

Experimental Procedure

Animals were allocated to treatment groups using randomisation tables.Each treatment group consisted of three animals, with 6 animals in thecontrol group. The test sample was dosed to young female rats with bodyweight 100–150 g at acclimatisation, for three consecutive days. Animalswere identified by means of metallic ear tags and KMnO₄ skin markingsfor easy identification. Animals were housed individually in standardrodent polycarbonate cages, and water and powdered commercial rodentpellets were available ad libitum. Water and food intakes were measuredand calculated for each day. In order to find the minimum effective doseof the test sample, five doses were tested. Treatment was by oralgavage, with the test sample suspended in potato starch.

The test substance was compound (1), a white granular powder preparedfrom an extract from plant material in accordance with the invention,and the measured quantity of the test sample was mixed with preparedpotato starch and dosed. Mixing with potato starch took placeimmediately before dosing on each day. Before withdrawal of the dosingvolume for each animal, the suspensions were mixed thoroughly using aVortex.

A range of five doses was tested, with a control group 15 receiving onlythe carrier substance. Doses were chosen on the basis of the effectsobserved in the aforedescribed Irwin Test and were:

-   Group 1: 0.00 mg/kg (Control Group)-   Group 2: 6.25 mg/kg-   Group 3: 12.50 mg/kg-   Group 4: 25.00 mg/kg-   Group 5: 37.50 mg/kg-   Group 6: 50.00 mg/kg    Results

Treatment did not affect the health of the animals during the studyperiod. Animals treated with the test sample in all dose groups, showeda significantly reduced mean body mass gain over the total study period,and animals in three of the five treatment groups actually lost bodymass.

Mean food intakes for all the treatment groups were reduced over thestudy period. Animals in the higher dose groups showed an increasedwater consumption.

Respiratory rate in none of the animals in any dose group wassignificantly effected.

Animals in all dose groups presented with friable livers at post-mortemexamination, but no macroscopic is pathology was observed.

Discussion

Data collected during the acclimatisation period confirmed that allanimals included in the experiment were healthy and body mass gain wascomparable between the animals.

The reduction, and in some animals even a loss, in body mass gain, incombination with the reduced food intake is strongly indicative ofsuppression of the appetite centre.

Reduced food intake and reduced body mass gain was experienced even withthe lowest dose group (6.25 mg/kg). Actual loss in body mass wasexperienced in the 12.50 mg/kg group.

It is important to note that the treatment groups all had an increasedwater consumption when feed consumption decreased (FIG. 2). This couldbe due to a diuretic effect of the test sample, or to stimulation of thethirst centre in the brain.

The fact that no respiratory suppression occurred as had been observedin the acute toxicity test referred to above, with the intraperitonealroute, is seen as a positive aspect. This could be due to reducedabsorption from the gastrointestinal tract, with consequent reducedbioavailability. The bioavailability at the oral doses tested was,however, sufficient for the test sample to be effective. The slightreduction in respiratory rate 1 hour post treatment in most groups couldbe ascribed to filling of the stomach with the dose volume andconsequent passivity of the animals.

The friable livers observed in the treatment groups could be due to achange in the energy metabolism secondary to the reduced food intake,causing increased fat metabolism and overload on the liver. If this wasindeed the case, these changes could possibly be regarded these changesas transitory which might recover with time after a steady state hadbeen reached, or after withdrawal of the test sample. The possibleeffect on the liver also needs further investigation.

Since this study was intended primarily as a screening test, smallgroups of test animals were used. This makes statistical interpretationof the data difficult, especially where individual animals react totallydifferently. However, the data indicates that the test sample hasappetite suppressive action, even at the lowest dose tested (6,25mg/kg). No clinical signs of respiratory suppression occurred at thedoses tested.

EXAMPLE 43

Harvested Hoodia plants received either from the natural environment orthrough a cultivation programme are first stored at 4° C. for a maximumof 48 hours. The plants are washed in tap water and thereafter slicedinto ±1 cm slices. The sliced pieces are all combined and then pressedthrough a hydraulic press at 300 bar pressure for a minimum of 0.5 hourper pressing. During the pressing the sap of the plant is collectedseparately. The sap is stored at −18° C. until further processing isrequired.

The sap is spray-dried under suitable conditions to obtain a freeflowing powder. The moisture content in the powder is preferably lessthan 5% after spray drying and, if necessary, it is further dried in avacuum oven or using a fluid bed drier.

Both the sap and the spray-dried material have been shown effective asan appetite suppressant in biological assays in rats.

Experimental

50 kg of Hoodia gordonii plants were washed with tap water andthereafter sliced into 1 cm slices. The sliced plants were then pressedthrough a hydraulic press at 300 bar for a minimum of 0.5 hour perbatch. The sap was collected and the mass was found to be 10 kg whenHoodia gordonii plants from the environment were used, and 20 kg whenHoodia gordonii plants from the cultivation programme was used. The sap(500 g) was spray-dried using the following conditions:

Flow rate 2.85 ml/min Inlet temperature 110° C. Outlet temperature 70°C. Chamber temperature 78° C.

The spray-dried powder obtained was a free flowing powder (22 g) with amoisture content of 6.9%.

The spray dried powder was analysed for active ingredient concentrationusing HPLC techniques. The concentration of the active was determined tobe 13 g/kg of spray dried powder.

HPLC Analysis Method

Eluant Acetonitrile: water (7:3), isocratic Column Reverse phase C-18 UVabsorbance 225 nm Flow rate 1 ml/min Injection volume 10 μlMethod

Spray-dried powder (10 mg) was dissolved in water (0.5 ml) andacetonitrile (0.5 ml) 10 μl of this solution was injected into the HPLCand the concentration of the active compound (1) was determined using astandard curve which was prepared from the pure compound (1).

EXAMPLE 44

The results of a study designed to assess the possible anorectic effectsof compound (1) in the rat are presented below. In the following, thesamples tested are pure sap (Sample 1), spray-dried sap (Sample 2) andactive moiety (Sample 3). Samples 1 and 2 are the sap and thespray-dried sap respectively, as described in Example 43 above. Sample 3is solvent-extracted compound (1) of ≧95% purity.

Sample 1 to 3 were each administered as a single oral dose to maleWistar rats. Two additional control groups received vehicle (distilledwater or DMSO). Orally administered fenfluramine (7.5 mg/kg) wasincluded as a reference standard.

Sample 1 (pure sap) administered orally, produced dose-dependentreductions in food consumption which were statistically significant atdoses of 1600 mg/kg and above when compared with vehicle-treatedcontrols. Concomitant reductions in bodyweight (or growth rate) werealso recorded. On the day of dosing, statistically significant increasesin water consumption were recorded at 3 hours post-dose (6400 and 10000mg/kg) and 6 hours post-dose (10000 mg/kg). Between 24 and 48 hourspost-dose, statistically significant reductions in water consumptionwere recorded at doses of 3200 mg/kg and above.

Sample 2 (spray-dried sap) administered orally at 76 mg/kg also producedstatistically significant reductions in food consumption and bodyweightwhen compared with vehicle-treated animals. No statistically significanteffects on water consumption were recorded.

Sample 3 (active moiety) produced statistically significant reductionsin food consumption at an oral dose of 5.0 mg/kg. No statisticallysignificant effects on bodyweights were produced by the active moietyalthough examination of the data revealed a slight delay in growth whencompared with vehicle-treated control animals. No statisticallysignificant effects on water consumption were recorded.

The reference standard, fenfluramine (7.5 mg/kg), produced statisticallysignificant reductions in food consumption at 6 and 24 hours post-dosewhen compared with the relevant vehicle-treated control group. Nostatistically significant effects on water consumption or bodyweightwere recorded.

No treatment-related effects on the livers were recorded.

TEST SUBSTANCE Identity Sample 1 (pure sap) Sample 2 (spray-dried sap)Sample 3 (active moiety) Appearance Brown liquid Powder White powderStorage conditions −20° C. in the dark Room temperature in the 4° C. inthe dark dark Purity Pure sap Pure spray-dried sap ≧95% VehicleDistilled water Distilled water Dimethylsulphoxide (DMSO)Experimental Procedure

Fifty-five male Wistar rats were used for the study.

Bodyweights, food consumption (food hopper weight) and water consumption(bottle weight) were recorded daily at the same time each day from theday of arrival until the termination of the study.

On Day 1, the rats received a single oral (gavage) dose according to thefollowing table:

Dose Group n Oral treatment (mg/kg) 1 5 Vehicle (distilled water) — 2 4Sample 1 (pure sap) 800 3 5 Sample 1 (pure sap) 1600 4 5 Sample 1 (puresap) 3200 5 5 Sample 1 (pure sap) 6400 6 5 Sample 1 (pure sap) 10000 7 5Sample 2 spray-dried sap 38 8 5 Sample 2 spray-dried sap 76 9 5 Sample 3(active moiety) 2.5 10 5 Sample 3 (active moiety) 5.0 11 3 Fenfluramine7.5 12 3 Vehicle (DMSO) —

Groups 1–8 were dosed using a constant dose volume of 10 ml/kg andgroups 9–12 were dosed using a dose volume of 1 ml/kg.

Food and water consumption were also measured at 1.3 and 6 hours afterdosing on Day 1.

Following the measurements of Day 8, the animals were killed by carbondioxide asphyxiation, and the livers excised and placed in 10% bufferedformalin, prior to histology.

Paraffin wax sections of each liver were taken at 4–5 μm and stainedwith haematoxylin and eosin. Additional sections were cut on a cryostatat 12 μm and stained for fat with Oil Red O (ORO).

Data Analysis

The post-dose food and water consumption measurements and bodyweights ateach time-point for the P57-treated animals were compared with those forthe relevant, similarly-treated vehicle control group using analysis ofvariance followed by Williams' test for comparisons with controls.

The data for the fenfluramine-treated animals was compared with that forthe vehicle-treated control group using Student's t test.

Results

The results are summarised in the tables.

Sample 1 (pure sap) administered orally produced marked, dose-relatedreductions in daily food consumption. The duration and amplitude ofthese reductions in food consumption were dose-dependent. At 24 hourspost-dose, Sample 1 (pure sap) produced statistically significantreductions in food consumption at doses of 1600 mg/kg and above whencompared with vehicle-treated controls. The highest dose of Sample 1(sap) (10000 mg/kg) produced statistically significant reductions infood consumption on a daily basis up to 5 days post-dose.

Sample 2 (spray-dried sap) and Sample 3 (active moiety) produced markedand statistically significant reductions in food consumption at oraldoses of 76 and 5.0 mg/kg respectively. In both cases the effects lasted48 hours post-dose.

The reference standard, fenfluramine (7.5 mg/kg, p.o.) producedstatistically significant reductions in food consumption at 6 and 24hours post-dose when compared with the relevant vehicle-treated controlgroup (Group 12).

Sample 2 (spray-dried sap) and Sample 3 (active moiety) produced nomarked, dose-related effects on water consumption. On the day of dosing,the pure sap produced statistically significant increases in waterconsumption at 3 hours post-dose (6400 and 10000 mg/kg) and 6 hourspost-dose (10000 mg/kg). Two days after dosing however, statisticallysignificant decreases in water consumption were recorded in animalsreceiving Sample 1 (sap) at 3200, 6400 and 10000 mg/kg. These reductionshowever, were not clearly dose-related and only occurred between 1 and 2days post-dose. The biological significance of these effects thereforeremains unclear.

Sample 1 (pure sap) produced dose-related, statistically significanteffects on bodyweights when compared with the vehicle-treated controlgroup (Group 1). When administered orally at doses of 3200 mg/kg andabove, Sample 1 (pure sap) produced statistically significant reductionsin bodyweight or decreased growth rates when compared withvehicle-treated animals. These effects were statistically significantfrom 48 hours post-dose until the end of the study.

Sample 2 (spray-dried sap) administered orally at 76 mg/kg also producedstatistically significant reductions in growth of the animals whencompared with the vehicle-treated control group (Group 1). These effectswere statistically significant between Days 3 (48 hours post-dose) and 5inclusive.

Although Sample 3 (active moiety) appeared to delay the growth of theanimals at the highest dose (5.0 mg/kg) when compared with the relevantvehicle-treated control group (Group 12), this effect was notstatistically significant.

Fenfluramine, (7.5 mg/kg) produced no marked or statisticallysignificant effects on water consumption or bodyweights when comparedwith the vehicle-treated control group (Group 12).

No treatment-related effects on the livers were recorded.

TABLE 1a Effects of oral administration on food consumption in the rat(daily pre-dose data) Group mean food consumption (g ± sd) between Days:Group Oral treatment Dose (mg/kg) −6–−5 −5–−4 −4–−3 −3–−2 −2–−1 1Vehicle (water) — 27.8 ± 1.54 24.2 ± 1.83 27.6 ± 3.67 28.3 ± 3.50 29.4 ±2.66 2 Sample 1 sap 800 28.3 ± 1.43 24.9 ± 0.82 27.7 ± 0.76 28.4 ± 1.5130.1 ± 0.27 3 Sample 1 sap 1600 29.0 ± 1.39 25.0 ± 2.16 27.4 ± 1.96 28.8± 0.61 29.5 ± 1.55 4 Sample 1 sap 3200 27.2 ± 2.33 25.1 ± 2.46 26.0 ±2.52 28.5 ± 2.29 27.6 ± 1.15 5 Sample 1 sap 6400 28.7 ± 1.64 25.3 ± 1.7327.3 ± 1.45 29.2 ± 1.09 30.3 ± 0.90 6 Sample 1 sap 10000 28.5 ± 2.3823.7 ± 2.73 26.0 ± 2.31 27.0 ± 3.50 28.7 ± 2.26 7 Sample 2 spray-dried38 28.1 ± 1.24 23.9 ± 1.79 24.5 ± 2.30 27.6 ± 1.61 28.5 ± 1.87 8 Sample2 spray-dried 76 28.7 ± 0.91 26.5 ± 1.55 27.1 ± 1.01 28.7 ± 1.99 28.9 ±1.37 9 Sample 3 active moiety 2.5 28.8 ± 1.49 26.4 ± 3.12 29.0 ± 1.9929.4 ± 1.76 29.5 ± 2.81 10 Sample 3 active moiety 5.0 28.3 ± 2.1  25.8 ±1.86 28.1 ± 2.65 28.0 ± 2.65 28.5 ± 3.03 11 Fenfluramine 7.5 29.1 ± 0.6625.3 ± 4.03 27.0 ± 1.53 30.8 ± 0.54 29.7 ± 2.84 12 Vehicle (DMSO) — 27.9± 1.8  26.7 ± 2.11 28.7 ± 1.99 28.1 ± 4.06 30.5 ± 2.54 sd Standarddeviation

TABLE 1b Effects of oral administration on food consumption in the rat(daily post-dose data) Group mean food consumption (g ± sd) betweenDays: Group Oral treatment Dose (mg/kg) 1–2 2–3 3–4 4–5 1 Vehicle(water) — 29.5 ± 3.15 29.6 ± 2.84 30.6 ± 3.49 31.8 ± 3.21 2 Sample 1 sap800 26.1 ± 0.98 29.3 ± 1.49 30.7 ± 1.15 30.9 ± 0.60 3 Sample 1 sap 1600 22.6 ± 3.17** 26.9 ± 2.06 30.9 ± 2.54 30.9 ± 1.22 4 Sample 1 sap 3200 20.1 ± 1.39**  19.0 ± 1.88**  22.8 ± 1.77** 28.0 ± 3.14 5 Sample 1 sap6400  18.2 ± 4.18**  14.8 ± 1.75**  18.4 ± 0.97**  22.4 ± 3.01** 6Sample 1 sap 10000  15.1 ± 2.98**  12.4 ± 2.61**  16.0 ± 3.15**  19.7 ±4.31** 7 Sample 2 spray-dried 38 25.6 ± 2.85 27.3 ± 0.95 30.3 ± 2.0631.0 ± 2.13 8 Sample 2 spray-dried. 76  24.2 ± 3.25*  25.2 ± 3.24* 29.9± 1.85 30.2 ± 2.28 9 Sample 3 active moiety 2.5 26.8 ± 3.33 29.1 ± 3.4331.7 ± 3.08 34.0 ± 2.95 10 Sample 3 active moiety 5.0  22.1 ± 2.19†† 21.0 ± 3.07†† 27.6 ± 5.26 30.5 ± 3.33 11 Fenfluramine 7.5  22.4 ±3.19^(†) 31.9 ± 0.84 32.7 ± 2.50 33.0 ± 2.55 12 Vehicle (DMSO) — 29.9 ±3.36 30.6 ± 4.43 30.1 ± 4.17 32.4 ± 5.26 Group mean food consumption (g± sd) between Days: Group Oral treatment Dose (mg/kg) 5–6 6–7 7–8 1Vehicle (water) — 30.7 ± 2.24 31.7 ± 3.03 32.9 ± 3.18 2 Sample 1 sap 80033.3 ± 1.69 32.7 ± 0.80  40.1 ± 13.40 3 Sample 1 sap 1600 34.1 ± 1.3633.7 ± 1.69 33.8 ± 1.61 4 Sample 1 sap 3200 31.4 ± 2.82 32.3 ± 2.91 33.0± 3.01 5 Sample 1 sap 6400 26.9 ± 2.81 31.0 ± 2.31 32.0 ± 2.34 6 Sample1 sap 10000  22.6 ± 5.70* 30.1 ± 4.79 32.6 ± 5.90 7 Sample 2 spray-dried38 31.8 ± 1.63 31.1 ± 1.94 31.8 ± 2.45 8 Sample 2 spray-dried. 76 31.2 ±2.26 32.3 ± 1.44 33.1 ± 0.61 9 Sample 3 active moiety 2.5 34.4 ± 4.3233.1 ± 4.11 34.8 ± 3.71 10 Sample 3 active moiety 5.0 33.0 ± 3.16 32.4 ±3.25 33.0 ± 3.84 11 Fenfluramine 7.5 30.4 ± 0.23 32.7 ± 1.90 32.4 ± 1.6012 Vehicle (DMSO) — 31.8 ± 3.08 32.8 ± 3.98 33.3 ± 3.76 sd Standarddeviation Groups 2–8 were compared with vehicle Group 1: *p< 0.05, **p <0.01 Groups 9–11 were compared with vehicle Group 12: †p < 0.05, ††p <0.01

TABLE 2a Effects of oral administration on water consumption in the rat(daily pre-dose data) Group mean water consumption (g ± sd) betweenDays: Group Oral treatment Dose (mg/kg) −6–−5 −5–−4 −4–−3 −3–−2 −2–−1 1Vehicle (water) — 40.9 ± 4.61 34.8 ± 4.15 37.6 ± 5.63 33.5 ± 7.42 32.2 ±6.32 2 Sample 1 sap 800 38.6 ± 1.96 37.1 ± 9.74 36.4 ± 4.81 28.1 ± 1.8330.4 ± 4.75 3 Sample 1 sap 1600  43.4 ± 10.53 35.9 ± 3.84 38.4 ± 4.5631.1 ± 4.47 36.5 ± 5.39 4 Sample 1 sap 3200 40.1 ± 5.58 33.3 ± 3.01 37.3± 4.46 31.3 ± 3.48 31.7 ± 3.18 5 Sample 1 sap 6400 43.8 ± 8.57 36.3 ±9.02 35.4 ± 8.18 34.0 ± 6.62 35.1 ± 5.72 6 Sample 1 sap 10000 37.4 ±5.34 32.7 ± 3.35 33.2 ± 4.86 29.0 ± 5.11 32.2 ± 3.27 7 Sample 2spray-dried 38 40.0 ± 4.36 35.8 ± 4.92 34.7 ± 3.20 30.2 ± 1.88 31.4 ±2.98 8 Sample 2 spray-dried 76 38.6 ± 1.98 37.0 ± 1.96 48.8 ± 21.5 31.6± 4.56  39.0 ± 17.27 9 Sample 3 active moiety 2.5 42.0 ± 6.70 37.0 ±5.05 34.1 ± 3.16 28.0 ± 2.58 31.6 ± 3.12 10 Sample 3 active moiety 5.040.9 ± 4.48 34.2 ± 3.00 32.7 ± 1.26 28.2 ± 1.65 33.1 ± 4.82 11Fenfluramlne 7.5 47.0 ± 5.3  35.5 ± 7.49 34.7 ± 3.73 30.9 ± 2.12 31.6 ±2.80 12 Vehicle (DMSO) — 43.3 ± 5.67 34.5 ± 4.97 35.2 ± 4.34 28.3 ± 4.6431.4 ± 6.44 sd Standard deviation

TABLE 2b Effects of oral administration on water consumption in the rat(daily post-dose data) Group mean water consumption (g ± sd) betweenDays: Group Oral treatment Dose (mg/kg) 1–2 2–3 3–4 4–5 1 Vehicle(water) — 34.9 ± 5.45 36.9 ± 6.06 38.0 ± 7.59 37.2 ± 6.18 2 Sample 1 sap800 30.9 ± 3.77 34.4 ± 8.12  38.2 ± 13.71  35.9 ± 13.51 3 Sample 1 sap1600 29.2 ± 1.66 31.7 ± 5.35  41.3 ± 11.21 34.6 ± 4.10 4 Sample 1 sap3200 35.9 ± 5.88  26.2 ± 2.66* 30.5 ± 2.44 34.1 ± 4.80 5 Sample 1 sap6400  33.4 ± 12.04  27.4 ± 8.13*  32.6 ± 10.67  35.4 ± 10.78 6 Sample 1sap 10000  31.7 ± 12.74  28.5 ± 8.85* 32.4 ± 8.87 36.6 ± 6.50 7 Sample 2spray-dried 38 36.0 ± 6.02 34.5 ± 1.79 38.2 ± 7.16 39.6 ± 7.09 8 Sample2 spray-dried 76  45.0 ± 19.03  39.1 ± 16.59  46.9 ± 18.34 35.9 ± 3.40 9Sample 3 active moiety 2.5 32.2 ± 4.01  36.1 ± 12.42  38.3 ± 11.71  41.5± 16.60 10 Sample 3 active moiety 5.0 33.9 ± 2.40 31.5 ± 8.12 35.1 ±3.82 37.7 ± 5.99 11 Fenfluramine 7.5 34.1 ± 3.60 37.2 ± 1.48 36.7 ± 3.9233.8 ± 2.89 12 Vehicle (DMSO) — 40.7 ± 9.10 33.8 ± 9.37 32.9 ± 7.07 35.2 ± 11.49 Group mean water consumption (g ± sd) between Days: GroupOral treatment Dose (mg/kg) 5–6 6–7 7–8 1 Vehicle (water) — 37.7 ± 5.5435.3 ± 2.86 36.5 ± 5.85 2 Sample 1 sap 800  39.5 ± 11.20 28.8 ± 1.2231.8 ± 5.58 3 Sample 1 sap 1600  48.1 ± 12.27 37.8 ± 7.28 36.9 ± 9.28 4Sample 1 sap 3200  45.8 ± 18.54  51.0 ± 35.21  42.6 ± 13.88 5 Sample 1sap 6400 45.2 ± 8.72 36.2 ± 6.72 35.9 ± 9.58 6 Sample 1 sap 10000  40.7± 11.51 38.0 ± 6.66 37.5 ± 6.21 7 Sample 2 spray-dried 38 42.7 ± 9.74 45.6 ± 17.15 46.1 ± 9.49 8 Sample 2 spray-dried 76  41.9 ± 12.37 36.9 ±8.47 38.1 ± 8.93 9 Sample 3 active moiety 2.5 34.7 ± 7.57 33.0 ± 4.2035.3 ± 8.70 10 Sample 3 active moiety 5.0 39.5 ± 7.78  37.4 ± 11.07 37.8± 6.42 11 Fenfluramine 7.5 33.7 ± 5.43 32.1 ± 1.93 33.6 ± 2.50 12Vehicle (DMSO) — 33.8 ± 9.82 32.3 ± 7.44 32.0 ± 7.22 sd Standarddeviation Groups 2–8 were compared with vehicle Group 1: *p < 0.05Groups 9–11 were compared with vehicle Group 12 (no significances)

TABLE 3a Effects of oral administration on bodyweight in the rat (dailypre-dose data) Group mean bodyweight (g ± sd on Day: Group Oraltreatment Dose (mg/kg) −5 −4 −3 −2 −1 1 Vehicle (water) — 130.9 ± 5.56150.7 ± 5.37 157.3 ± 5.29 168.1 ± 6.20 177.5 ± 6.70 2 Sample 1 sap 800131.6 ± 4.34 150.1 ± 4.84 158.5 ± 4.35 169.6 ± 4.99 177.7 ± 4.10 3Sample 1 sap 1600 130.1 ± 4.3  148.6 ± 6.59 156.7 ± 6.38 167.5 ± 6.04176.6 ± 6.37 4 Sample 1 sap 3200 130.8 ± 6.19 147.7 ± 7.56 154.4 ± 8.06165.2 ± 8.43 175.8 ± 9.10 5 Sample 1 sap 6400 132.6 ± 7.01 151.3 ± 7.23158.4 ± 8.50 169.0 ± 8.79 178.1 ± 7.75 6 Sample 1 sap 10000 132.3 ± 6.75151.8 ± 9.08 157.3 ± 9.37  167.1 ± 10.41  175.4 ± 10.90 7 Sample 2spray-dried 38 131.7 ± 8.28 149.0 ± 5.85 156.2 ± 5.81 166.7 ± 5.54 175.6± 8.42 8 Sample 2 spray-dried 76 130.0 ± 6.99 146.1 ± 6.00 155.9 ± 6.59166.0 ± 6.87 175.1 ± 6.55 9 Sample 3 active moiety 2.5 132.6 ± 7.63148.9 ± 8.51 157.3 ± 8.91 169.8 ± 8.96 179.4 ± 8.71 10 Sample 3 activemoiety 5.0 133.5 ± 6.45 150.5 ± 9.55 158.8 ± 8.48 171.0 ± 7.72 179.0 ±9.20 11 Fenfluramine 7.5 133.2 ± 9.21 152.7 ± 9.09 160.0 ± 9.82 170.0 ±9.15  182.8 ± 10.21 12 Vehicle (DMSO) — 129.1 ± 3.17 147.3 ± 4.37 155.0± 6.29 166.0 ± 5.91 174.8 ± 8.26 sd Standard deviation

TABLE 3b Effects of oral administration on bodyweight in the rat (dailypost-dose data) Group mean bodyweight (g ± sd) on Day: Group Oraltreatment Dose (mg/kg) Pre-dose (1) 2 3 4 5 1 Vehicle (water) — 185.4 ±7.77 192.6 ± 7.16 202.0 ± 10.17 211.2 ± 7.98 220.2 ± 10.35 2 Sample 1sap 800 186.0 ± 4.90 187.0 ± 4.55 198.5 ± 4.20  206.8 ± 5.91 214.8 ±4.65  3 Sample 1 sap 1600 185.0 ± 6.67 186.0 ± 8.28 193.2 ± 6.42  204.0± 6.40 212.4 ± 5.81  4 Sample 1 sap 3200 181.8 ± 9.18 184.6 ± 8.88 186.2± 8.67*  189.8 ± 9.99**  199.2 ± 9.34** 5 Sample 1 sap 6400 186.6 ± 7.96185.6 ± 6.39  183.8 ± 6.87**  185.2 ± 9.18**  191.2 ± 7.89** 6 Sample 1sap 10000  182.8 ± 12.22  181.4 ± 14.06  179.8 ± 15.85**   180.6 ±13.85**  185.6 ± 11.28** 7 Sample 2 spray-dried 38 183.4 ± 8.11 185.8 ±9.23 195.8 ± 7.79  205.6 ± 9.79 214.4 ± 9.61  0 Sample 2 spray-dried 76180.6 ± 6.47 183.4 ± 7.57 188.6 ± 6.73*  198.2 ± 8.50* 206.0 ± 9.43* 9Sample 3 active moiety 2.5 188.2 ± 9.42  191.2 ± 11.15 200.0 ± 11.25 209.6 ± 12.28 219.6 ± 12.95 10 Sample 3 active moiety 5.0  186.4 ±10.02 192.0 ± 9.93 192.4 ± 9.84   201.0 ± 11.27 209.4 ± 12.70 11Fenfluramine 7.5 190.3 ± 9.71  190.3 ± 10.97 197.7 ± 7.37  207.7 ± 7.23217.7 ± 10.69 12 Vehicle (DMSO) — 183.3 ± 8.33  190.3 ± 10.26 199.0 ±10.82  207.7 ± 12.66 215.7 ± 14.05 Group mean bodyweight (g ± sd) onDay: Group Oral treatment Dose (mg/kg) 6 7 8 1 Vehicle (water) — 227.2 ±10.26 235.8 ± 11.82 242.8 ± 11.97 2 Sample 1 sap 800 222.8 ± 4.99  231.5± 3.70  240.0 ± 3.65  3 Sample 1 sap 1600 223.0 ± 6.33  232.6 ± 7.70 240.4 ± 6.66  4 Sample 1 sap 3200  210.6 ± 10.21**  219.0 ± 11.29* 228.4 ± 12.18* 5 Sample 1 sap 6400 201.0 ± 6.89   213.0 ± 6.96**  222.0± 7.94** 6 Sample 1 sap 10000  192.2 ± 10.99** 203.4 ± 11.68  212.4 ±11.35** 7 Sample 2 spray-dried 38 222.6 ± 9.34  231.4 ± 10.62 239.6 ±11.46 0 Sample 2 spray-dried 76 214.0 ± 9.51  222.0 ± 9.49  232.2 ±9.68  9 Sample 3 active moiety 2.5 229.4 ± 13.69 238.4 ± 14.50 247.0 ±14.35 10 Sample 3 active moiety 5.0 219.8 ± 11.86 228.2 ± 12.28 236.0 ±13.95 11 Fenfluramine 7.5 224.3 ± 10.12 234.3 ± 12.70 243.3 ± 9.24  12Vehicle (DMSO) — 222.3 ± 14.84 230.7 ± 15.95 239.0 ± 17.35 sd Standarddeviation Groups 2–8 were compared with vehicle Group 1: *p < 0.05, **p< 0.01 Groups 9–11 wore compared with vehicle Group 12 (nosignificances)Histopathology Report

Histological examination was restricted to the liver. Notreatment-related changes were detected for Sample 1 (liquid), Sample 2(spray-dried sap), Sample 3 (active moiety), fenfluramine or the DMSOcontrol group.

The findings recorded were of a similar incidence in control and treatedgroups.

TABLE Microscopic pathology incidence summary Group 1 Group 2 Group 3Group 4 Group 5 Group 6 0 800 1600 3200 6400 10000 Sex: Males mg/kgmg/kg mg/kg mg/kg mg/kg mg/kg Males on study 5 4 5 5 5 5 Animalscompleted 5 4 5 5 5 5 Liver 5 Examined 5 4 5 5 5 0 No abnormalitiesdetected 0 0 1 2 3 3 Parenchymal inflammatory cell foci (Total) 0 1 0 00 3 Minimal 0 1 0 0 0 1 Hepatocyte hypertrophy - centrilobular (Total) 00 0 0 0 1 Minimal 0 0 0 0 0 0 Extramedullary haemopoiesis (Total 2 0 0 00 0 Minimal 2 0 0 0 0 0 Hepatocyte necrosis - focal (Total) 1 0 0 0 0 0Minimal 1 0 0 0 0 0 Portal lymphoid infiltration (Total) 3 4 4 3 2 2Minimal 3 4 4 3 2 2 Eosinophilic hepatocytes - focal (Total) 1 0 0 0 0 0Minimal 1 0 0 0 0 0 Portal fibrosis (Total) 0 0 1 0 0 0 Minimal 0 0 1 00 0 Liver (ORO stain) Examined 5 4 5 5 5 5 No abnormalities detected 2 32 4 3 3 Hepatocyte fat - centrilobular (Total) 3 1 2 1 2 2 Minimal 3 1 21 2 2 Hepatocyte fat - periportal (Total) 0 0 1 0 0 0 Minimal 0 0 1 0 00 Group 7 Group 8 Group 9 Group 10 Group 11 Group 12 38 76 2.5 5 7.5 0Sex: Males mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg Males on study 5 5 5 5 33 Animals completed 5 5 5 5 3 3 Liver Examined 5 5 5 5 3 3 Noabnormalities detected 2 2 0 1 0 2 Parenchymal inflammatory cell foci(Total) 0 0 0 0 0 1 Minimal 0 0 0 0 0 1 Hepatocyte necrosis - focal(Total) 0 0 1 0 0 0 Minimal 0 0 1 0 0 0 Portal lymphoid infiltration(Total) 3 3 5 4 3 1 Minimal 3 3 5 4 3 1 Portal leucocytes (Total) 0 0 10 0 0 Minimal 0 0 1 0 0 0 Liver (ORO stain) Examined 5 5 5 5 3 3 Noabnormalities detected 5 3 3 3 2 2 Hepatocyte fat - centrilobular(Total)0 2 2 2 1 1 Minimal 0 2 2 2 1 0

EXAMPLE 45

A further bioassay, which employed the same test samples as described inExample 44, is described below. Animals in this study received arestricted diet i.e. animals only received food between 12:00 and 3:00pmdaily. This is different from all other biological assays conducted thusfar, whereby food was available to the rats at lib. Animals wereacclimatised over a seven day period (days −7 to −1), dosing took placefrom day 0 to day 6 at 9:00am by oral gavage. The recovery period wasfrom days 7 to day 13. Dosage groups are described in Table 1 below. Itshould be noted that the actual control group is labelled Group 09.Group 5 is a controlled group which received a diet equivalent to thatof Group 4. The purpose of this group was to evaluate the effect arestricted diet has on the lives of the animals.

Results

The results generated during the study showed that the acclimatizationperiod was too short. Rats feed mainly during the night and the suddenchange to a restricted access to feed for 3 hours during day-time,resulted in low daily intakes. The daily intake of feed was stillincreasing in most groups at the end of the acclimatization period whendosing with the test items started. As a result of this, the effect ofthe test materials did not significantly affect the food intake of therats during the period of dosing.

The mean body masses for the different groups for day −7 to −1 and days0 to 6 are shown in the Table D1 and Table D2. The effect of thedifferent dosages of the sap and spray-dried sap is shown in theaccompanying graphs as % change in body mass day 0 to 7 (FIG. 5), and %change in body mass day −7 to 7 (FIG. 6). The loss in body mass isclearly dose-related especially with the higher dosages.

The histopathological examination of the livers did not show anysignificant pathology in the groups receiving the test items.

Food

Food consumption was measured daily, during acclimatization and duringthe study. Food was available for a 3 hour feeding period daily,starting at 12:00 and ending at 15:00. The animals were fasted for theremainder of the time. Animals in Group 5 received a measured quantityfood on Day 1, equivalent to the average food consumption of Group 4 onDay 0. This controlled feeding pattern for Group 5, as determined fromthe average food consumption of Group 4 from the previous day, wasfollowed for Days 1–7.

Water

Water was provided in standard containers. Water (Magalies Water BoardTap Water, suitable for human consumption) was available ad libitum.Water consumption was measured once daily, at the same time each day,after food consumption determination.

Acclimatization

The animals were acclimatized for seven days before the start of thestudy, during which time food and water consumption were determined asdescribed above. The body masses were determined on a daily basis duringthis time.

Study Design and Procedures

TABLE 1 STUDY DESIGN GROUP TEST NUMBERS DOSE TEST ITEM 01 6♂ 001–006 100mg/kg Frozen sap 02 6♂ 007–012 400 mg/kg Frozen sap 03 6♂ 013–018 1600mg/kg Frozen sap 04 6♂ 019–024 3200 mg/kg Frozen sap 05 6♂ 025–030CONTROL Elga Option 4 Purified Water 06 6♂ 031–036 2.2 mg/kg Spray-driedsap 07 6♂ 037–042 8.8 mg/kg Spray-dried sap 08 6♂ 043–048 35 mg/kgSpray-dried sap 09 6♂ 049–054 CONTROL Elga Option 4 Purified WaterRoute of Administration

The test items were administered on a daily basis for seven days, usingan intra-gastric needle. Animals were fasted for 18 hours prior to theitem administration (starting at 09:00).

Duration of Treatment

Animals were treated for seven consecutive days (from Day 0–Day 6).Three animals of each group were sacrificed 24 hours after the lastdosing (Day 7). The remaining three animals were sacrificed 7 days afterthe last treatment (Day 13). This procedure was followed for all thegroups except for Group 5 where three animals were sacrificed 24 hoursafter the last controlled feeding (Day 8), the remaining three animalswere sacrificed 7 days after the last treatment (Day 13).

Body Masses

Body masses were determined daily, at approximately the same time eachday for the duration of the study, including during the acclimatizationperiod.

Euthanasia

Three animals of each group were sacrificed 24 hours after the lastdosing (Day 7).

The remaining three animals were sacrificed 7 days after the lasttreatment. This procedure was followed for all the groups except forGroup 5 where three animals were sacrificed 24 hours after the lastcontrolled feeding (Day 8), the remaining three animals were sacrificed7 Days after the last treatment (Day 13). The animals were euthanased atthe end of the study period with CO₂ gas.

Ophthalmoscopic Examinations

Ophthalmoscopic examinations, using an ophthalmoscope, were done priorto the first adminstration of the test item and at termination, in allanimals in all groups.

Macroscopic Pathology

A full post mortem examination was performed on every animal which waseuthanased at the end of the study period.

Histopathology

Histopathological examination was performed on the liver of each of theanimals.

TABLE D.1 MEAN BODY MASSES/GROUP/WEEK Mean body masses (g) & Standarddeviation Group Oral treatment Dose (mg/kg) Day −7 Day −6 Day −5 Day −401 Sample 1 (Sap) 100 203.38 ± 95.39 197.13 ± 90.63 192.75 ± 89.49188.62 ± 86.75 02 Sample 1 (Sap) 400 192.53 ± 65.60 183.92 ± 61.20178.25 ± 59.37 173.17 ± 58.10 03 Sample 1 (Sap) 1600 149.25 ± 54.80142.87 ± 51.89 136.85 ± 52.17 132.37 ± 49.64 04 Sample 1 (Sap) 3200224.15 ± 80.70 214.45 ± 77.25 207.10 ± 76.38 201.82 ± 75.42 05 ElgaOption 4 — 214.55 ± 74.90 204.85 ± 72.41 198.57 ± 71.79 193.48 ± 68.49purified water (control) 06 Sample 2 (Spray-dried sap) 2.2 208.65 ±65.74 199.37 ± 62.49 193.18 ± 61.18 188.25 ± 60.89 07 Sample 2(Spray-dried sap) 8.8 256.95 ± 77.55 246.02 ± 73.67 237.47 ± 73.53232.62 ± 71.73 08 Sample 2 (Spray-dried sap) 35 194.37 ± 43.74 185.83 ±42.70 177.53 ± 41.10 172.05 ± 40.13 09 Elga Option 4 — 171.52 ± 69.81162.67 ± 62.68 154.95 ± 61.83 151.38 ± 59.48 purified water (control)Mean body masses (g) & Standard deviation Group Oral treatment Dose(mg/kg) Day −3 Day −2 Day −1 01 Sample 1 (Sap) 100 184.95 ± 84.80 182.48± 83.47 182.25 ± 82.57 02 Sample 1 (Sap) 400 170.82 ± 57.42 168.25 ±58.40 169.37 ± 59.25 03 Sample 1 (Sap) 1600 131.50 ± 49.50 129.67 ±48.89 131.12 ± 48.22 04 Sample 1 (Sap) 3200 198.25 ± 74.82 194.83 ±75.34 196.77 ± 74.56 05 Elga Option 4 — 192.40 ± 67.48 190.87 ± 67.39190.15 ± 65.24 purified water (control) 06 Sample 2 (Spray-dried sap)2.2 186.22 ± 59.98 184.55 ± 58.86 185.97 ± 58.76 07 Sample 2(Spray-dried sap) 8.8 229.78 ± 71.76 228.07 ± 69.88 228.45 ± 68.81 08Sample 2 (Spray-dried sap) 35 170.10 ± 39.49 167.25 ± 37.61 168.00 ±38.83 09 Elga Option 4 — 149.63 ± 57.66 148.30 ± 57.12 149.07 ± 56.01purified water (control)

TABLE D.2 MEAN BODY MASSES/GROUP/WEEK Mean body masses (g) & Standarddeviation Group Oral treatment Dose (mg/kg) Day 0 Day 1 Day 2 Day 3 01Sample 1 (Sap) 100 183.87 ± 83.33 175.83 ± 81.82 175.72 ± 79.05 175.48 ±77.54 02 Sample 1 (Sap) 400 173.45 ± 60.73 164.58 ± 58.52 164.75 ± 58.37166.22 ± 57.69 03 Sample (Sap) 1600 134.38 ± 46.01 129.20 ± 44.74 127.53± 43.20 127.20 ± 41.36 04 Sample (Sap) 3200 199.60 ± 75.16 196.38 ±73.96 192.20 ± 71.20 189.05 ± 69.11 05 Elga Option 4 — 194.27 ± 67.46187.93 ± 65.48 181.97 ± 65.01 177.53 ± 64.73 purified water (control) 06Sample 2 (Spray-dried sap) 2.2 189.07 ± 60.15 181.52 ± 58.99 181.48 ±57.79 184.42 ± 55.64 07 Sample 2 (Spray-dried sap) 8.8 230.28 ± 69.32221.55 ± 68.02 220.17 ± 66.63 221.80 ± 63.88 08 Sample 2 (Spray-driedsap) 35 169.10 ± 38.40 164.42 ± 38.03 162.50 ± 36.81 162.75 ± 36.36 09Elga Option 4 — 151.02 ± 55.45 146.55 ± 53.77 148.10 ± 52.67 149.70 ±52.05 purified water (control) Mean body masses (g) & Standard deviationGroup Oral treatment Dose (mg/kg) Day 4 Day 5 Day 6 01 Sample 1 (Sap)100 175.53 ± 76.20 177.95 ± 73.99 178.43 ± 72.68 02 Sample 1 (Sap) 400166.55 ± 57.79 169.93 ± 57.47 171.77 ± 57.29 03 Sample (Sap) 1600 126.70± 39.19 128.00 ± 39.22 128.07 ± 38.66 04 Sample (Sap) 3200 186.57 ±66.29 186.05 ± 67.45 185.68 ± 65.73 05 Elga Option 4 — 174.73 ± 61.08172.85 ± 58.63 171.45 ± 56.79 purified water (control) 06 Sample 2(Spray-dried sap) 2.2 185.75 ± 55.29 189.35 ± 54.66 189.68 ± 53.70 07Sample 2 (Spray-dried sap) 8.8 222.82 ± 63.56 224.82 ± 62.38 224.90 ±62.05 08 Sample 2 (Spray-dried sap) 35 162.52 ± 36.93 164.30 ± 37.69164.22 ± 37.18 09 Elga Option 4 — 152.58 ± 50.37 155.82 ± 49.91 157.85 ±49.70 purified water (control)

TABLE D.3 MEAN BODY MASSES/GROUP/WEEK (CONTINUED) Dose Mean body masses(g) & Standard deviation Group Oral treatment (mg/kg) Day 7 Day 8 Day 9Day 10 01 Sample 1 (Sap) 100 185.38 ± 72.64 234.73 ± 62.44 236.73 ±62.39 234.07 ± 62.09 (GHA I 35A) 02 Sample 1 (Sap) 400 178.83 ± 58.24225.63 ± 13.05 277.13 ± 14.18 227.10 ± 14.03 (GHA I 35A) 03 Sample 1(Sap) 1600 132.22 ± 37.08 133.80 ± 55.17 135.23 ± 455.74 134.53 ± 54.96(GHA I 35A) 04 Sample 1 (Sap) 3200 188.57 ± 66.14 199.63 ± 61.07 198.90± 57.48 198.70 ± 54.55 (GHA 9 35A) 05 Elga Option 4 — 173.97 ± 54.29172.98 ± 52.06 157.80 ± 58.62 158.87 ± 57.76 purified water (control) 06Sample 2 (Spray-dried sap) 2.2 196.00 ± 53.09 190.27 ± 27.78 190.27 ±29.54 192.60 ± 29.09 (GHA I 59) 07 Sample 2 (Spray-dried sap) 8.8 231.30± 61.91 177.27 ± 24.48 178.17 ± 23.79 180.67 ± 25.04 (GHA I 59) 08Spray-dried sap 35 167.48 ± 36.75 164.90 ± 22.54 166.63 ± 23.08 168.43 ±22.66 (GHA I 59) 09 Elga Option 4 — 165.50 ± 49.27 193.73 ± 22.37 196.87± 21.86 198.07 ± 21.02 purified water (control) Dose Mean body masses(g) & Standard deviation Group Oral treatment (mg/kg) Day 11 Day 12 Day13 01 Sample 1 (Sap) 100 236.33 ± 62.31 239.07 ± 60.24 238.43 ± 59.85(GHA I 35A) 02 Sample 1 (Sap) 400 229.43 ± 16.97 234.93 ± 18.35 236.20 ±15.97 (GHA I 35A) 03 Sample 1 (Sap) 1600 138.30 ± 53.03 139.30 ± 51.10142.80 ± 49.51 (GHA I 35A) 04 Sample 1 (Sap) 3200 194.73 ± 52.78 194.93± 50.78 197.93 ± 51.57 (GHA 9 35A) 05 Elga Option 4 — 160.80 ± 57.67163.40 ± 56.27 167.80 ± 58.49 purified water (control) 06 Sample 2(Spray-dried sap) 2.2 194.73 ± 29.68 196.97 ± 29.04 198.60 ± 30.18 (GHAI 59) 07 Sample 2 (Spray-dried sap) 8.8 182.03 ± 25.31 185.10 ± 24.60189.73 ± 23.58 (GHA I 59) 08 Spray-dried sap 35 171.67 ± 24.42 174.90 ±25.70 178.57 ± 23.58 (GHA I 59) 09 Elga Option 4 — 199.83 ± 20.21 204.93± 18.65 207.13 ± 18.22 purified water (control)

TABLE 1 HISTOLOGICAL EVALUATION OF LIVER SECTIONS FROM MALE RATS Sample1 Animal no Hepatic lesions GROUP 1: 100 mg/kg Sample 1 Day 7 01 NPL 02NPL 03 NPL C1 + Day 13 04 NPL MLC 05 FHS1 + 06 NPL GROUP 2: 400 mg/kgSample 1 07 FHS1 + 08 NPL C1 + 09 NPL Day 13 10 DHS1 + 11 NPL 12 DHS1 +GROUP 3: 1600 mg/kg Sample 1 Day 7 13 NPL 14 NPL 15 NPL Day 13 16 NPL 17DHS1 + 18 NPL GROUP 4: 3200 mg/kg Sample 1 19 NPL 20 NPL 21 NPL Day 1322 DHS1 + 23 FHS1 + 24 NPL GROUP 5: CONTROL: ELGA OPTION 4 PURIFIEDWATER: RESTRICTED FOOD INTAKE GROUP 5: Control: Elga option 4 purifiedwater Day 7 25 NPL MLC 26 NPL 27 NPL Day 13 28 DHS1 + 29 DHS1 + 30 NPLLegend: C = Congestion DHS = Diffuse hydropic cell swelling FHS = Focalhydropic cell swelling NPL = No parenchymal lesions MLC = Minimallymphocytic cuffing 1 + = mild 2 + = moderate 3 + = severe

TABLE 2 HISTOLOGICAL EVALUATION OF LIVER SECTIONS FROM MALE RATS Sample2 Animal no Hepatic lesions GROUP 6: 2.2 mg/kg Sample 2 Day 7 31 NPL 32NPL MLC 33 FHS1 + Day 13 34 NPL 35 DHS1 + 36 NPL GROUP 7: 8.8 mg/kgSample 2 37 NPL 38 NPL 39 NPL C1 + Day 13 40 DHS1 + 41 NPL 42 MLC FHS1 +GROUP 8: 35 mg/kg Sample 2 Day 7 43 NPL 44 NPL 45 NPL Day 13 46 NPL 47NPL C1 + 48 MLC FHS1 + GROUP 9: CONTROL: ELGA OPTION 4 PURIFIED WATERGROUP 9: Control: Elga option 4 purified water Day 7 49 NPL 50 NPL 51FHS1 + Day 13 52 DHS1 + 53 NPL 54 FHS1 + Legend: C = Congestion DHS =Diffuse hydropic cell swelling FHS = Focal hydropic cell swelling NPL =No parenchymal lesions MLC = Minimal lymphocytic cuffing 1 + = mild 2 += moderate 3 + = severe

No specific lesions were recorded in the liver sections from theexperimental rats which received the frozen sap as well as thespray-dried sap that could be attributed to the oral adminstration ofthe abovementioned chemicals. The hydropic cell swelling recorded inboth control and experimental rats may indicate normal metabolic cellswelling and anoxic changes. Minimal foci of lymphocytic perivascularcuffing were found in some animals and is most likely an incidentalobservation. In a few rats congestion of mild degree is present in thehepatic sinusoids and should be regarded as an incidental observation.

An important feature of the invention shown by the results of this studyis that no tolerance to any of the samples developed over the testperiod. This may provide considerable benefit, particularly in relationto the use of the compounds and compositions of the invention in thetreatment of obesity.

While the compounds and compositions of the invention have primarilybeen described in relation to their properties as appetite suppressants,it should be noted that this expression—“appetite suppressant” — is usedherein to denote activity which tends to limit appetite and/or increasethe sense of satiety, and thus tends to reduce total calorific intake;this in turn tends to counteract obesity. Accordingly, this inventionextends to a method of treating, preventing or combating obesity in ahuman or non-human animal which comprises administering to said human ornon-human animal an obesity treating, preventing or combating amount ofa compound of formula (2). A preferred embodiment of this aspect of theinvention utilises a composition or extract containing a compound offormula (1).

The term “animal” as used herein extends to, but is not restricted to,companion animals, e.g. household pets and domesticated animals;non-limiting examples of such animals include cattle, sheep, ferrets,swine, camels, horses, poultry, fish, rabbits, goats, dogs and cats.

As an anorectic agent or in the treatment or prevention of obesity in ahuman, a compound of formula (2), preferably of formula (1), or thecomposition defined in any one of claims 9 and 25–31 hereafter, isadvantageously administered to said human in a dosage amount of fromabout 0.01 mg/kg/day to about 10 mg/kg/day. A preferred dosage range is0.05 mg/kg/day to 0.5 mg/kg/day. When using the spray dried powder formof the extract of this invention, a preferred dosage range is 0.1mg/kg/day to 20 mg/kg/day; especially preferred is 0.5 mg/kg/day to 5mg/kg/day.

1. A method of suppressing appetite in a human or animal, comprisingadministering to said human or animal an appetite-suppressant effectiveamount of a compound having the structural formula (2)

wherein: R=alkyl; R₁=H, alkyl, tigloyl, benzoyl, or any other organicester group; R₂=H, or one or more 6-deoxy carbohydrates, or one or more2,6-dideoxy carbohydrates, or glucose molecules, or combinationsthereof; and the broken lines indicate the optional presence of afurther bond between C4–G5 or C5–C6.
 2. The method as claimed in claim1, wherein said compound has the structural formula (1)


3. A method of suppressing appetite in a human or animal, comprisingadministering to said human or animal an appetite-suppressant effectiveamount of an extract from a plant of the genus Trichocaulon or the genusHoodia, wherein said extract comprises a compound having the structuralformula (2)

wherein: R=alkyl; R₁=H, alkyl, tigloyl, benzoyl, or any other organicester group; R₂=H, or one or more 6-deoxy carbohydrates, or one or more2,6-dideoxy carbohydrates, or glucose molecules, or combinationsthereof; and the broken lines indicate the optional presence of afurther bond between C4–C5 or C5–C6.
 4. The method as claimed in claim3, wherein said compound has the structural formula (1)


5. The method as claimed in claim 3 or claim 4, wherein said extract isprepared by a process comprising treating material from said plant witha solvent to extract at least said compound present in said materialinto said solvent; separating said solvent from said plant material; andremoving said solvent to provide an extract having appetite-suppressantactivity and comprising said compound.
 6. The method as claimed in claim5, wherein substantially all non-active impurities have been removedfrom said extract having appetite-suppressant activity.
 7. The method asclaimed in claim 4, wherein said extract is prepared by a processcomprising pressing material from said plant to separate sap from saidplant material that is solid, wherein said sap has appetite-suppressantactivity and comprises said compound; and recovering said sap free ofsaid solid plant material to form said extract.
 8. The method as claimedin claim 7, wherein substantially all non-active impurities have beenremoved from said sap having appetite suppressant activity.
 9. Themethod as claimed in claim 3 or claim 4, wherein said plant of the genusTrichocaulon is selected from the species Trichocaulon piliferum andTrichocaulon officinale and said plant of the genus Hoodia is selectedfrom the species Hoodia currorii, Hoodia gordonii and Hoodia lugardii.10. The method as claimed in claim 5, wherein the process furthercomprises extracting said extract with an additional and differentsolvent to increase the concentration of said compound in said extract.11. The method as claimed in claim 10, wherein the solvent is methylenechloride, water, methanol, hexane, ethyl acetate or mixtures thereof.12. The method as claimed in claim 10, wherein said different solvent ismethylene chloride, water, methanol, hexane, ethyl acetate or mixturesthereof.
 13. A method as claimed in claim 4, further comprisingsubjecting said extract to chromatographic separation to increase theconcentration of said compound in said extract.
 14. The method asclaimed in claim 13, wherein said chromatographic separation employschloroform, methanol, ethyl acetate, hexane or mixtures thereof as aneluant.
 15. The method as claimed in claim 4, wherein said extract is afree flowing powder.
 16. The method as claimed in claim 4, wherein saidextract is a spray-dried extract of said plant.
 17. The method asclaimed in claim 4, wherein said extract is a freeze-dried extract ofsaid plant.
 18. The method as claimed in claim 4, wherein said extractis a vacuum-dried extract of said plant.
 19. The method as claimed inclaim 4, wherein said compound is isolated or purified from said plant.20. The method as claimed in claim 4, wherein said compound is isolatedor purified from said extract.
 21. The method as claimed in claim 4,wherein a pharmaceutical composition comprising said compound is in unitdosage form.
 22. The method as claimed in claim 4, wherein said plant isselected from Hoodia currorii, Hoodia gordonii and Hoodia lugardii. 23.The method as claimed in claim 4, wherein said plant is selected fromHoodia gordonii and Hoodia lugardii.
 24. The method as claimed in claim4, wherein said plant is Hoodia gordonii.
 25. The method as claimed inclaim 1, wherein a pharmaceutical composition comprising said compoundand a pharmaceutical excipient, carrier or diluent is administered tosaid human or animal.
 26. The method as claimed in claim 2, wherein acomposition comprising said compound and a pharmaceutical excipient,carrier or diluent is administered to said human or animal.
 27. Themethod as claimed in claim 4, wherein a pharmaceutical compositioncomprising said extract from the plant of the genus Trichocaulon or thegenus Hoodia and a pharmaceutical excipient, carrier or diluent isadministered to said human or animal.
 28. The method as claimed in claim1, wherein a foodstuff comprising said compound is administered to saidhuman or animal.
 29. A method as claimed in claim 2, wherein a foodstuffcomprising said compound is administered to said human or animal. 30.The method as claimed in claim 4, wherein a foodstuff comprising saidextract from said plant of the genus Trichocaulon or the genus Hoodia isadministered to said human or animal.
 31. The method as claimed in claim1, wherein a beverage comprising said compound is administered to saidhuman or animal.
 32. The method as claimed in claim 2, wherein abeverage comprising said compound is administered to said human oranimal.
 33. The method as claimed in claim 4, wherein a beveragecomprising said extract from said plant of the genus Trichocaulon or thegenus Hoodia is administered to said human or animal.
 34. The method asclaimed in any one of claims 1–4, wherein said human or animal is ahuman.
 35. The method as claimed in claim 34, wherein said human is anobese human.
 36. A method of reducing total calorific intake of a humanor animal, comprising administering to said human or animal anappetite-suppressant effective amount of a compound having thestructural formula (2)

wherein: R=alkyl; R₁=H, alkyl, tigloyl, benzoyl, or any other organicester group; R₂=H, or one or more 6-deoxy carbohydrates, or one or more2,6-dideoxy carbohydrates, or glucose molecules, or combinationsthereof; and the broken lines indicate the optional presence of afurther bond between C4–C5 or C5–C6.
 37. The method as claimed in claim36, wherein said compound has the structural formula (1)


38. A method of reducing total calorific intake of a human or animal,comprising administering to said human or animal an appetite-suppressanteffective amount of an extract from a plant of the genus Trichocaulon orthe genus Hoodia, wherein said extract comprises a compound having thestructural formula (2)

wherein: R=alkyl; R₁=H, alkyl, tigloyl, benzoyl, or any other organicester group; =R₂=H, or one or more 6-deoxy carbohydrates, or one or more2,6-dideoxy carbohydrates, or glucose molecules, or combinations thereofand the broken lines indicate the optional presence of a further bondbetween C4–C5 or C5–C6.
 39. The method as claimed in claim 38, whereinsaid compound has the structural formula (1)


40. The method as claimed in claim 38 or claim 39, wherein said extractis prepared by a process comprising treating material from said plantwith a solvent to extract at least said compound present in saidmaterial into said solvent; separating said solvent from said plantmaterial; and removing said solvent to provide an extract havingappetite-suppressant activity and comprising said compound.
 41. Themethod as claimed in claim 40, wherein substantially all non-activeimpurities have been removed from said extract havingappetite-suppressant activity.
 42. The method as claimed in claim 39,wherein said extract is prepared by a process comprising pressingmaterial from said plant to separate sap from said plant material thatis solid, wherein said sap has appetite-suppressant activity andcomprises said compound; and recovering said sap free of said solidplant material to form said extract.
 43. The method as claimed in claim42, wherein substantially all non-active impurities have been removedfrom said sap having appetite suppressant activity.
 44. The method asclaimed in claim 38 or claim 39, wherein said plant of the genusTrichocaulon is selected from the species Trichocaulon piliferum andTrichocaulon officinale and said plant of the genus Hoodia is selectedfrom the species Hoodia currorii, Hoodia gordonii and Hoodia lugardii.45. The method as claimed in claim 40, wherein the process furthercomprises extracting said extract with an additional and differentsolvent to increase the concentration of said compound in said extract.46. The method as claimed in claim 40, wherein the solvent is methylenechloride, water, methanol, hexane, ethyl acetate or mixtures thereof.47. The method as claimed in claim 45, wherein said different solvent ismethylene chloride, water, methanol, hexane, ethyl acetate or mixturesthereof.
 48. A method as claimed in claim 40, further comprisingsubjecting said extract to chromatographic separation to increase theconcentration of said compound in said extract.
 49. The method asclaimed in claim 48, wherein said chromatographic separation employschloroform, methanol, ethyl acetate, hexane or mixtures thereof as aneluant.
 50. The method as claimed in claim 39, wherein said extract is afree-flowing powder.
 51. The method as claimed in claim 39, wherein saidextract is a spray-dried extract of said plant.
 52. The method asclaimed in claim 39, wherein said extract is a freeze-dried extract ofsaid plant.
 53. The method as claimed in claim 39, wherein said extractis a vacuum-dried extract of said plant.
 54. The method as claimed inclaim 39, wherein said compound is isolated or purified from said plant.55. The method as claimed in claim 39, wherein said compound is isolatedor purified from said extract.
 56. The method as claimed in claim 39,wherein a composition comprising said compound is in unit dosage form.57. The method as claimed in claim 39, wherein said plant is selectedfrom Hoodia currorii, Hoodia gordonii and Hoodia lugardii.
 58. Themethod as claimed in claim 39, wherein said plant is selected fromHoodia gordonii and Hoodia lugardii.
 59. The method as claimed in claim39, wherein said plant is Hoodia gordonii.
 60. The method as claimed inclaim 36, wherein a pharmaceutical composition comprising said compoundand a pharmaceutical excipient, carrier or diluent is administered tosaid human or animal.
 61. The method as claimed in claim 37, wherein apharmaceutical composition comprising said compound and a pharmaceuticalexcipient, carrier or diluent is administered to said human or animal.62. The method as claimed in claim 39, wherein a pharmaceuticalcomposition comprising said extract from the plant of the genusTrichocaulon or the genus Hoodia and a pharmaceutical excipient, carrieror diluent is administered to said human or animal.
 63. The method asclaimed in claim 36, wherein a foodstuff comprising said compound isadministered to said human or animal.
 64. A method as claimed in claim37, wherein a foodstuff comprising said compound is administered to saidhuman or animal.
 65. The method as claimed in claim 39, wherein afoodstuff comprising said extract from said plant of the genusTrichocaulon or the genus Hoodia is administered to said human oranimal.
 66. The method as claimed in claim 36, wherein a beveragecomprising said compound is administered to said human or animal. 67.The method as claimed in claim 37, wherein a beverage comprising saidcompound is administered to said human or animal.
 68. The method asclaimed in claim 4, wherein a beverage comprising said extract from saidplant of the genus Trichocaulon or the genus Hoodia is administered tosaid human or animal.
 69. The method as claimed in any one of claims36–39, wherein said human or animal is a human.
 70. The method asclaimed in claim 69, wherein said human is an obese human.